Anti-ATF3 antibody [EPR22610-19] - ChIP Grade

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

ab254268 staining ATF3 in wild-type Hap1 cells (top panel) and ATF3 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab254268 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling ATF3 with ab254268 at 1/500 dilution (1.14 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human placenta (PMID/ 28947613). Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (human monocytic leukemia monocyte) treated with 80nM Phorbol 12-myristate 13-acetate (PMA) for 16h, then together with 1μg/ml lipopolysaccharides (LPS) for 8h (Red) / Untreated control (Green) cells labelling ATF3 with ab254268 at 1/600 compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^®488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Immunohistochemical analysis of paraffin-embedded Human tissue labeling ATF3 with ab254268 at 1/500 dilution (1.14 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in Reed-Sternberg (HRS) cells of human (PMID : 16263788) is observed. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labelling ATF3 with ab254268 at 1/100 (5.7 ug/ml) dilution, followed by ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary antibody at 1/1000 (5.7 ug/ml) dilution (Green). Confocal image showing nuclear staining in THP-1 cells treated with Phorbol 12-myristate 13-acetate (80 nM) for 16 h, then along with lipopolysaccharides (1ug/ml) for 8 h. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^® 488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

• ChIP

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ChIP - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol. Cells were fixed with EGS for 30min, then formaldehyde for 10min. The ChIP was performed with 25 μg of chromatin, 5 μg of ab254268 (red), and 20 μl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach). Primers and probes are located in the first kb of the transcribed region.

• IP

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Immunoprecipitation - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

ATF3 was immunoprecipitated from 0.35 mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate 10ug with ab254268 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254268 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate 10ug.

Lane 2 : ab254268 IP in RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab254268 in RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2h whole cell lysate.

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

Exposure time : 6 seconds.

All lanes:

Immunoprecipitation - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268)

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Lanes 1-2 : Merged signal (red and green). Green - ab254268 observed at 21 kDa. Red - loading control ab8245 observed at 37 kDa.

ab254268 Recombinant Anti-ATF3 antibody [EPR22610-19] was shown to specifically react with ATF3 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266955 (knockout cell lysate ab257075) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab254268 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

ATF3 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human ATF3 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-atf3-knockout-a549-cell-line-ab266955'>ab266955</a>)

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Blocking and dilution buffer : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID : 24973221; 24062788).

All lanes:

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Lane 2:

THP-1 treated with 80nM TPA overnight, then treated with 1ug/ml lipopolysaccharide (LPS) for 8 hours, whole cell lysate at 20 µg

Lane 3:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Lane 4:

RAW264.7 treated with 1ug/ml lipopolysaccharide (LPS) for 2 hours, 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

Predicted band size: 21 kDa

Observed band size: 21 kDa

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Exposure time: 70s

• WB

Lab

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Lanes 1-2 : Merged signal (red and green). Green - ab254268 observed at 21 kDa. Red - loading control ab8245 observed at 37 kDa.

ab254268 Recombinant Anti-ATF3 antibody [EPR22610-19] was shown to specifically react with ATF3 in wild-type HCT116 cells. Loss of signal was observed when knockout cell line ab266872 (knockout cell lysate ab257074) was used. Wild-type and ATF3 knockout samples were subjected to SDS-PAGE. ab254268 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

ATF3 knockout HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human ATF3 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-atf3-knockout-hct116-cell-line-ab266872'>ab266872</a>)

Predicted band size: 21 kDa

Observed band size: 21 kDa

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• WB

Lab

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

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

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

ATF3 has a low expression level in some cell lines and tissues, but is increased under treatment (PMID : 8622660, PMID : 22053207, PMID : 20018623, PMID : 29940414).

All lanes:

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Lane 3:

Raw264.7 (Mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

MEF (Mouse embryonic fibroblast (immortalized)) whole cell 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 1/20000 dilution

Predicted band size: 21 kDa

Observed band size: 21 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

Blocking and dilution buffer : 5% NFDM/TBST.

Negative control : Daudi (PMID : 19136462).

The molecular weight observed is consistent with what has been described in the literature (PMID : 18692824).

All lanes:

Western blot - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (ab254268) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Lane 4:

Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate at 20 µg

Lane 5:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 6:

ATF3 knockout HAP1 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

Predicted band size: 21 kDa

Observed band size: 21 kDa

false

Exposure time: 26s

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-ATF3 antibody [EPR22610-19] - ChIP Grade (AB254268)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL. 2.5X10^5 of Human ATF3 knockout HeLa cell line (ab264908) or Human wild-type HeLa cell line (ab255448) were used along with 5µg of Anti-ATF3 antibody (ab254268). Assay Quality Control was conducted using 5µg Anti-CTCF (ab188408) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.