Anti-Smad4 antibody [EPR22589-112]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed Smad4 KO HAP1 (Smad4 knockout human chronic myelogenous leukemia near-haploid cell line, Left) / WT HAP1 (human chronic myelogenous leukemia near-haploid cell line, Right) cell line labeling Smad4 with ab230815 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^� 488, ab150077) was used as secondary antibody at 1/2000 dilution.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Immunofluorescent analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Smad4 with ab230815 at a 1/100 dilution (green). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Confocal image showing mainly nuclear staining in HeLa cell line treated with TGF-beta (10ng/ml) for 1 h. AlexaFluor^®488 Goat anti-Rabbit (ab150077) was used secondary antibody at 1/1000 dilution, this was also used on its own as a control. DAPI was used as a nuclear counterstain (blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) was used as a counterstain at 1/200 dilution.

• IP

Unknown

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Smad4 was immunoprecipitated from 0.35mg of HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab230815 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab230815 in HeLa whole cell lysate

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 30 seconds

Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.

All lanes:

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (ab230815)

Predicted band size: 60 kDa

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• IP

Unknown

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Smad4 was immunoprecipitated from 0.35mg of NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab230815 at 1/30 dilution. Western Blot was performed from the immunoprecipitate using ab230815 at 1/1000 dilution (0.5 μg/ml).

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : NIH/3T3 whole cell lysate 10μg (Input).

Lane 2 : ab230815 IP in NIH/3T3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab230815 in NIH/3T3 whole cell lysate

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 10 seconds

Lysate was freshly prepared and IP test was done immediately to avoid protein degradation. Lysate incubation time was shortened from overnight to 2h.

All lanes:

Immunoprecipitation - Anti-Smad4 antibody [EPR22589-112] (ab230815)

Predicted band size: 60 kDa

false

• WB

Unknown

Western blot - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure Time : Lanes 1-6 : 15 secs; Lanes 7-8 : 114 secs; Lane 9 : 3 mins.

ab230815 was shown to specifically react with Smad4 in wild-type HAP1 cells as signal was lost in Smad4 knockout cells. Wild-type and Smad4 knockout samples were subjected to SDS-PAGE. ab230815 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution

Lane 1:

Smad4 knockout HAP1 whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

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

Lane 6:

Human fetal lung tissue lysate at 20 µg

Lane 7:

Mouse embryo tissue lysate at 20 µg

Lane 8:

Mouse lung tissue lysate at 20 µg

Lane 9:

Rat lung 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

Predicted band size: 60 kDa

Observed band size: 60 kDa

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• WB

Supplier Data

Western blot - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 15 secs; Lane 2 : 7.75 secs.

All lanes:

Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution

Lane 1:

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

Lane 2:

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 1/100000 dilution

Predicted band size: 60 kDa

Observed band size: 60 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad4 antibody [EPR22589-112] (AB230815)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with hTGF-β1 (10 ng/mL 1 h) and 5 µg of ab230815 [EPR22589-112]. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad4 antibody [EPR22589-112] (AB230815)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with hTGF-β1 (10 ng/mL 1 h) and 5 µg of ab230815 [EPR22589-112]. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-Smad4 antibody [EPR22589-112] (AB230815)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with hTGF-β1 (10 ng/mL 1 h) and 5 µg of ab230815 [EPR22589-112]. 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.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Unknown

Western blot - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution

Lane 1:

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

Lane 2:

NIH/3T3 (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 1/100000 dilution

Predicted band size: 60 kDa

false

Exposure time: 48s

• WB

CiteAb

Western blot - Anti-Smad4 antibody [EPR22589-112] (AB230815)

Smad4 western blot using anti-Smad4 antibody [EPR22589-112] ab230815. Publication image and figure legend from Song, Y., Wang, Z., et al., 2020, J Cell Mol Med, PubMed 33124760.

ab230815 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab230815 please see the product overview.

Effects of DEK shRNA on Smad, MAPK and PI3K/AKT signalling pathways in BEAS-2B cells. (A-E) The expressions of TGF-β, DEK mad2/3 and Smad4, ERK1/2, p38, JNK, and PI3K, AKT, mTOR were detected by Western blot. The relative density of each protein was calculated. Data were shown as mean ± SD (n = 3). NC, negative control cells; ovDEK, cells transfected with DEK; shNC, cells transfected with scrambled shRNA; shDEK, cells transfected with DEK shRNA. *p < 0.05, vs control group. #p < 0.05, ovDEK vs TGF-β1 + NC group; shDEK vs TGF-β1 + shNC group

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