Anti-GAPDH antibody [EPR16891] - Loading Control

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocyte of rat spleen is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized HeLa (human cervix adenocarcinoma) cells labeling GAPDH with ab181602 at dilution of 1/500. The secondary antibody used was Alexa Fluor^® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/400. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/500) was used to stain tubulin along with ab150120 (AlexaFluor^®594 goat anti-mouse secondary, 1/500). The negative controls are shown in the bottom middle and right hand panels- for negative control 1 primary antibody (ab181602; 1/500) and secondary antibody (ab150120; 1/500) was used. For negative control 2 primary antibody (ab7291; 1/500) and secondary antibody (ab150077; 1/400) was used.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling GAPDH with ab181602 at 1/180 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded human transitional cell carcinoma of bladder tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasmic and nucleus staining on the tumor cells of transitional cell carcinoma of human bladder is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Dnmt3a was immunoprecipitated from 0.35 mg Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab323708 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323708 at 1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-Dnmt3a antibody [EPR29184-89] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a>) at 1/1000 dilution

Lane 1:

(Input) Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> at 1/30 IP in Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> in parental HeLa whole cell lysate at 10 µg

Lane 4:

(Input) DNMT3A knockout HeLa whole cell lysate at 10 µg

Lane 5:

<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> at 1/30 IP in DNMT3A knockout HeLa whole cell lysate at 10 µg

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr29184-89-ab323708'>ab323708</a> in DNMT3A knockout HeLa whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 130 kDa,36 kDa

false

Exposure time: 24s

• IP

Lab

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

STT3A was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) whole cell lysate with ab320831 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab320831 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Immunoprecipitation, ab320831 was shown to bind specifically to STT3A. Target of interest was enriched at 60 kDa in wild-type HEK-293T cell lysates (lane 2) with no signal observed at this size in STT3A knockout cell line (lane 5) (lane 5, knockout cell line ab266320 / knockout cell lysate ab259164).
The bands above 60kDa are likely to be aggregation, and are absent in the KO samples. Unlike the Western Blot data, these samples have been boiled. ab181602 was used as a GAPDH loading control.

All lanes:

Immunoprecipitation - Anti-STT3A antibody [EPR29178-10] (<a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a>) at 1/1000 dilution

Lane 1:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

Parental HEK-293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in Parental HEK-293T whole cell lysate

Lane 4:

STT3A KO HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 5:

Immunoprecipitation - Human STT3A knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-stt3a-knockout-hek-293t-cell-line-ab266320'>ab266320</a>)

Lane 6:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/stt3a-antibody-epr29178-10-ab320831'>ab320831</a> in STT3A KO HEK-293T whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 60 kDa

false

Exposure time: 84s

• IP

Supplier Data

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

GAPDH was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab181602 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab181602 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : HeLa whole cell extract.

Lane 2 : PBS instead of HeLa whole cell extract.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602)

Predicted band size: 36 kDa

Observed band size: 36 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and IL1r1-KO homozygous mice (Strain ID : T005977).

All lanes:

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IL1 Receptor I/IL-1R-1 antibody [EPR22198-36] (<a href='/en-us/products/primary-antibodies/il1-receptor-i-il-1r-1-antibody-epr22198-36-ab229051'>ab229051</a>) at 1/1000 dilution

Lane 1:

Wild-type mouse spleen tissue lysate (female) at 20 µg

Lane 2:

IL1r1 knockout mouse spleen tissue lysate (female) at 20 µg

Lane 3:

Wild-type mouse spleen tissue lysate (male) at 20 µg

Lane 4:

IL1r1 knockout mouse spleen tissue lysate (male) at 20 µg

Lane 5:

Wild-type mouse thymus tissue lysate (female) at 20 µg

Lane 6:

IL1r1 knockout mouse thymus tissue lysate (female) at 20 µg

Lane 7:

Wild-type mouse thymus tissue lysate (male) at 20 µg

Lane 8:

IL1r1 knockout mouse thymus tissue lysate (male) at 20 µg

Secondary

All lanes:

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 81 kDa,36 kDa

false

Exposure time: 3min

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling GAPDH with ab181602 at 1/2000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus and cytoplasmic staining on lymphocytes of mouse spleen is observed. Counter stained with Hematoxylin.

Negative control : Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low/negative samples : SW480 (PMID : 31324782), HeLa (PMID : 1463733)

All lanes:

Western blot - Anti-Prothrombin antibody [RM1252] (<a href='/en-us/products/primary-antibodies/prothrombin-antibody-rm1252-ab322113'>ab322113</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 72 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : spleen.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28223357; PMID : 26977393).

The identity of the higher MW band at approximately 300 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-ACSL5 antibody [EPR30222-556] (<a href='/en-us/products/primary-antibodies/acsl5-antibody-epr30222-556-ab327086'>ab327086</a>) at 1/1000 dilution

Lane 1:

Human small intestine tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Lane 3:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 70 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : HeLa

To minimize protein degradation, the fresh lysates were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes1-2 : 70 seconds, lane 3 : 48 seconds

All lanes:

Western blot - Anti-Bile Acid Receptor/FXR antibody [EPR29793-33] (<a href='/en-us/products/primary-antibodies/bile-acid-receptor-fxr-antibody-epr29793-33-ab325493'>ab325493</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 80 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 80 µg

Lane 3:

Huh7 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 80 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa,55 kDa,36 kDa

true

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with overexpressed human SLC34A1 or SLC34A3 by western blot.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (75-100KDa).

All lanes:

Western blot - Anti-SLC34A2 antibody [EPR30528-645] (<a href='/en-us/products/primary-antibodies/slc34a2-antibody-epr30528-645-ab325747'>ab325747</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 5 µg

Lane 2:

293T cells transfected with human SLC34A2 expression vector containing a myc-His-tag® whole cell lysate at 5 µg

Lane 3:

293T cells transfected with human SLC34A1 expression vector containing a myc-His-tag® whole cell lysate at 5 µg

Lane 4:

293T cells transfected with human SLC34A3 expression vector containing a myc-His-tag® whole cell lysate at 5 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 75-150 kDa,36 kDa,75-100 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : skeletal muscle(PMID : 16446356)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes1-2 : 70 seconds, lane 3 : 48 seconds

All lanes:

Western blot - Anti-Bile Acid Receptor/FXR antibody [EPR29793-33] (<a href='/en-us/products/primary-antibodies/bile-acid-receptor-fxr-antibody-epr29793-33-ab325493'>ab325493</a>) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 50 kDa,55 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-KDM6B / JMJD3 antibody [EPR28325-184] (<a href='/en-us/products/primary-antibodies/kdm6b-jmjd3-antibody-epr28325-184-ab324740'>ab324740</a>) at 1/1000 dilution

Lane 1:

Untreated SW620 (human colorectal adenocarcinoma epithelial cell) whole cell fresh lysate at 20 µg

Lane 2:

SW620 treated with 80 nM TPA for 8 hours whole cell fresh lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 200 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Western blot : Anti-COIL antibody [Pdelta] (ab11822) staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab11822 was shown to bind specifically to COIL. A band was observed at 68 kDa in wild-type HeLa cell lysates with no signal observed at this size in COIL knockout cell line. To generate this image, wild-type and COIL knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Coilin antibody [Pdelta] (<a href='/en-us/products/primary-antibodies/coilin-antibody-pdelta-ab11822'>ab11822</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

COIL knockout HeLa cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Observed band size: 68 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : A549

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-MafB antibody [EPR29843-56] (<a href='/en-us/products/primary-antibodies/mafb-antibody-epr29843-56-ab326360'>ab326360</a>) at 1/1000 dilution

Lane 1:

SK-N-MC (human brain epithelial cell) whole cell lysate at 20 µg

Lane 2:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 45 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1,000,000 dilution.

Lanes 1 - 2:

Western blot - Anti-Choline Acetyltransferase antibody [EPR13024(B)] (<a href='/en-us/products/primary-antibodies/choline-acetyltransferase-antibody-epr13024b-ab181023'>ab181023</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (<a href='/en-us/products/primary-antibodies/ddddk-tag-binds-to-flag-tag-sequence-antibody-epr20018-251-ab205606'>ab205606</a>) at 1/1000 dilution

Lanes 1 and 3:

293T cells transfected with an empty vector containing a flag tag whole cell lysate at 20 µg

Lanes 2 and 4:

293T cells transfected with a full-length human ChAT expression vector containing a flag-tag whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 82 kDa,36 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

ab284389 detected a non-specific band at approximately 100 kDa.

Negative control : HEK-293T, HeLa.

All lanes:

Western blot - Anti-CD133 antibody [RM1029] (<a href='/en-us/products/primary-antibodies/cd133-antibody-rm1029-ab284389'>ab284389</a>) at 1/1000 dilution

Lanes 1 and 6:

Caco-2 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 7:

NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 3 and 8:

HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lanes 4 and 9:

HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lanes 5 and 10:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 120 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with overexpressed human EMP1 by western blot.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (20-50KDa); Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-EMP2 antibody [EPR30305-511] (<a href='/en-us/products/primary-antibodies/emp2-antibody-epr30305-511-ab326423'>ab326423</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 5 µg

Lane 2:

293T cells transfected with a human EMP1 expression vector containing a myc-His-tag® whole cell lysate at 5 µg

Lane 3:

293T cells transfected with a human EMP2 expression vector containing a myc-His-tag® whole cell lysate at 5 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 20-50 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1/200000) (36KDa).

All lanes:

Western blot - Anti-Wnt5a/b antibody [EPR29464-527] (<a href='/en-us/products/primary-antibodies/wnt5a-b-antibody-epr29464-527-ab325179'>ab325179</a>) at 1/1000 dilution

Lane 1:

Hela conditioned media at 50 µg

Lane 2:

HeLa whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 42 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, ab325532 was shown to bind specifically to Wnt5a. Target of interest was observed at 42 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in WNT5A knockout cell line (lane 2) (lane 2, knockout cell line ab264019 / knockout cell lysate ab264515).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Wnt5a antibody [EPR29464-509] (<a href='/en-us/products/primary-antibodies/wnt5a-antibody-epr29464-509-ab325532'>ab325532</a>) at 1/1000 dilution

Lane 1:

WI-38 (human fetal lung fibroblast) whole cell lysate at 80 µg

Lane 2:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 80 µg

Lane 3:

WNT5A knockout HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 80 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 42 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

False colour image of Western blot : Anti-beta III Tubulin antibody [2G10] staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody ab181602 loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab78078 was shown to bind specifically to beta III Tubulin. A band was observed at 50 kDa in wild-type HeLa cell lysates with no signal observed at this size in the beta III Tubulin knockout cell line. To generate this image, wild-type and beta III Tubulin knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat Anti-Mouse IgG H&L (IRDye^® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) (ab216777) at 1/10000 dilution.

All lanes:

Western blot - Anti-beta III Tubulin antibody [2G10] - Neuronal Marker (<a href='/en-us/products/primary-antibodies/beta-iii-tubulin-antibody-2g10-neuronal-marker-ab78078'>ab78078</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

TUBB3 knockout HeLa whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-sima343b-ab325355'>ab325355</a>) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa treated with 0.5mM CoCl2 for 6 hours whole cell lysate at 20 µg

Lane 3:

Untreated HeLa whole cell lysate at 20 µg

Lane 4:

HeLa treated with 0.5mM DFO for 24 hours whole cell lysate at 20 µg

Lane 5:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 treated with 100µM CoCl2 for 4 hours whole cell lysate at 20 µg

Lane 7:

Untreated NIH/3T3 whole cell lysate at 20 µg

Lane 8:

NIH/3T3 treated with 0.5mM CoCl2 for 24 hours, 10µM MG-132 was then added for last 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, ab325355 was shown to bind specifically to HIF-1 alpha. A band was observed at 110 kDa in wild-type HCT 116 cell lysates with whereas no signal observed at this size in HIF-1 alpha knockout cell line ab255394.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-HIF-1 alpha antibody [SIMA343B] (<a href='/en-us/products/primary-antibodies/hif-1-alpha-antibody-sima343b-ab325355'>ab325355</a>) at 1/1000 dilution

Lane 1:

Untreated HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HCT116 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Lane 3:

Untreated HIF-1 alpha knockout HCT116 whole cell lysate at 20 µg

Lane 4:

HIF-1 alpha knockout HCT116 treated with 0.5 mM DFO for 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 110 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28223357; PMID : 26977393)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa); Anti-TOMM20 antibody [EPR15581-54] - Mitochondrial Marker (ab186735) (1 : 1000) (16KDa).

All lanes:

Western blot - Anti-ACSL5 antibody [EPR30222-556] (<a href='/en-us/products/primary-antibodies/acsl5-antibody-epr30222-556-ab327086'>ab327086</a>) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) non-mitochondrial fraction at 20 µg

Lane 2:

HCT 116 mitochondrial fraction at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 70 kDa,36 kDa,16 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody specifically recognizes the HDGFL2 cryptic peptide.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (140KDa).

All lanes:

Western blot - Anti-HDGFL2 cryptic peptide antibody [EPR31252-542] (ab328148) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a his-tag, whole cell lysate at 5 µg

Lane 2:

293T cells transfected with a human HDGFL2 cryptic peptide expression vector containing a his-tag, whole cell lysate at 5 µg

Lane 3:

293T cells transfected with a human wild-type HDGFL2 expression vector containing a his-tag, whole cell lysate at 5 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 140 kDa,36 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : SH-SY5Y, MCF7, PC-3(PMID : 28223357).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28223357; PMID : 26977393)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-ACSL5 antibody [EPR30222-556] (<a href='/en-us/products/primary-antibodies/acsl5-antibody-epr30222-556-ab327086'>ab327086</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

KATO III (human stomach spherical) whole cell lysate at 20 µg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 70 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(24-30KDa).

All lanes:

Western blot - Anti-IL-31 antibody [EPR31053-655] (ab328153) at 1/1000 dilution

Lane 1:

Untreated 293T (human embryonic kidney epithelial cell) cells transfected with a human IL-31expression vector containing a his-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human IL-31 expression vector containing a his-tag, whole cell lysate treated with Protein Deglycosylation Peptide:N-glycosidase F (PNGase F) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 24-30 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : CTLL-2 and Jurkat (PMID : 19147785).

The molecular weight observed is consistent with what has been described in the literature (PMID : 26516065; PMID : 14530327)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CD19 antibody [RM2072] (<a href='/en-us/products/primary-antibodies/cd19-antibody-rm2072-ab322345'>ab322345</a>) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Daudi (human Burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 3:

Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 4:

NAMALWA (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 5:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 6:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 20 µg

Lane 7:

WEHI-231 (mouse B cell lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 8:

CTLL-2 (mouse T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60-120 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : HepG2 (PMID : 30790340).

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-ETS1 antibody [EPR21909] (<a href='/en-us/products/primary-antibodies/ets1-antibody-epr21909-ab220361'>ab220361</a>) at 1/1000 dilution

Lane 1:

KARPAS-299 (human anaplastic large cell lymphoma cell line) whole cell lysate at 20 µg

Lane 2:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 51 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, ab325540 was shown to bind specifically to NPR1. Target of interest was observed at 130 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in NPR1 knockout cell line (lane 2) (lane 2, knockout cell line ab265930 / knockout cell lysate ab258080).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 6 seconds, lane 3 : 180 seconds

All lanes:

Western blot - Anti-NPR-A/Atrial natriuretic peptide receptor 1 antibody [EPR30453-586] (<a href='/en-us/products/primary-antibodies/npr-a-atrial-natriuretic-peptide-receptor-1-antibody-epr30453-586-ab325540'>ab325540</a>) at 1/1000 dilution

Lane 1:

Parental HeLa(human cervical adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

Western blot - Human NPR1 (Natriuretic Peptide Receptor A/GC-A) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-npr1-natriuretic-peptide-receptor-a-gc-a-knockout-hela-cell-lysate-ab258080'>ab258080</a>) at 40 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 40 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 130 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

Negative control : A549 (human lung carcinoma epithelial cell) cells.

All lanes:

Western blot - Anti-DARC antibody [EPR5205] (<a href='/en-us/products/primary-antibodies/darc-antibody-epr5205-ab137044'>ab137044</a>) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 2:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 37-50 kDa,36 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : HepG2, U-2 OS, SUM149PT.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SLC34A2 antibody [EPR30528-645] (<a href='/en-us/products/primary-antibodies/slc34a2-antibody-epr30528-645-ab325747'>ab325747</a>) at 1/1000 dilution

Lane 1:

NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SUM149PT (human breast adenocarcinoma cell) whole cell lysate at 20 µg

Lane 4:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 75-100 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Low expression : HT-29 (PMID : 29628506).

The bands between 40 kDa and 60 kDa are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lane 1 : 8 seconds, Lanes 2-6 : 15 seconds

All lanes:

Western blot - Anti-PARIS antibody [EPR29119-192] (<a href='/en-us/products/primary-antibodies/paris-antibody-epr29119-192-ab325709'>ab325709</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 60 µg

Lane 2:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 60 µg

Lane 3:

SW620 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 60 µg

Lane 4:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 60 µg

Lane 5:

HCT 116 (human colorectal carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 60 µg

Lane 6:

HCT 116 transfected with siRNA specifically targeting ZNF746 whole cell lysate at 60 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 90 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with overexpressed human DLL3 or DLL4 by western blot.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(85, 30KDa); Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-DLL1 antibody [EPR29655-26] (<a href='/en-us/products/primary-antibodies/dll1-antibody-epr29655-26-ab327088'>ab327088</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human DLL1 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human DLL3 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human DLL4 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 85 kDa,30 kDa,36 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

In Western blot, Anti-DDDDK tag (Binds to FLAG^® tag sequence) antibody [EPR20018-251] (ab205606) 1/1000 dilution.

All lanes:

Western blot - Anti-beta IV Tubulin antibody [EPR16776] (<a href='/en-us/products/primary-antibodies/beta-iv-tubulin-antibody-epr16776-ab179509'>ab179509</a>) at 1/5000 dilution

Lane 1:

HEK293T (human embryonic kidney epithelial cell) cells transfected with an empty vector whole cell lysate at 20 µg

Lane 2:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 1 whole cell lysate at 20 µg

Lane 3:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 2A whole cell lysate at 20 µg

Lane 4:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 2B whole cell lysate at 20 µg

Lane 5:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 3 whole cell lysate at 20 µg

Lane 6:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 4A whole cell lysate at 20 µg

Lane 7:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 4B whole cell lysate at 20 µg

Lane 8:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 5 whole cell lysate at 20 µg

Lane 9:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 6 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa,36 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : Human skeletal muscle, mouse heart.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CD19 antibody [RM2072] (<a href='/en-us/products/primary-antibodies/cd19-antibody-rm2072-ab322345'>ab322345</a>) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 20 µg

Lane 2:

Human spleen tissue lysate at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Lane 4:

Mouse lymph node tissue lysate at 20 µg

Lane 5:

Mouse spleen tissue lysate at 20 µg

Lane 6:

Mouse heart tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60-120 kDa,36 kDa

false

Exposure time: 103s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

In Western blot, Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) 1 : 1000 dilution.

All lanes:

Western blot - Anti-beta I Tubulin antibody [EPR16778] (<a href='/en-us/products/primary-antibodies/beta-i-tubulin-antibody-epr16778-ab179511'>ab179511</a>) at 1/5000 dilution

Lane 1:

HEK293T (human embryonic kidney epithelial cell) cells transfected with an empty vector whole cell lysate at 20 µg

Lane 2:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 1 whole cell lysate at 20 µg

Lane 3:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 2A whole cell lysate at 20 µg

Lane 4:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 2B whole cell lysate at 20 µg

Lane 5:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 3 whole cell lysate at 20 µg

Lane 6:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 4A whole cell lysate at 20 µg

Lane 7:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 4B whole cell lysate at 20 µg

Lane 8:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 5 whole cell lysate at 20 µg

Lane 9:

HEK293T (human embryonic kidney epithelial cell) + OE- Tubulin beta- 6 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa,36 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : Raji

Performed under reducing conditions.

In Western blot, ab325607 was shown to bind specifically to TMEM106B. Target of interest was observed at 40 and 30 kDa in wild-type A549 cell lysates (lane 1) with no signal observed at this size in TMEM106B knockout cell line (lane 2, knockout cell line ab273711 / knockout cell lysate ab273769).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 39709600).

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

The identity of the bands higher than 150 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-TMEM106B antibody [EPR30569-44] - C-terminal (<a href='/en-us/products/primary-antibodies/tmem106b-antibody-epr30569-44-c-terminal-ab325607'>ab325607</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human TMEM106B knockout A549 cell lysate (<a href='/en-us/products/cell-lysates/human-tmem106b-knockout-a549-cell-lysate-ab273769'>ab273769</a>) at 20 µg

Lane 3:

AsPC-1 (human Pancreas epithelial cell) whole cell lysate at 20 µg

Lane 4:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 6:

3T3-L1 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 30 kDa,40 kDa,36 kDa

true

Exposure time: 103s

• WB

Lab

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (AB181602)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000) (53KDa); Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Lipoprotein lipase antibody [EPR31098-680] (<a href='/en-us/products/primary-antibodies/lipoprotein-lipase-antibody-epr31098-680-ab328031'>ab328031</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human Lipoprotein lipase expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 53 kDa,36 kDa

false

Exposure time: 180s