All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution

Lane 1 : Smad4 knockout HAP1 whole cell lysate
Lane 2 : Wild-type HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 5 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 6 : Human fetal lung tissue lysate
Lane 7 : Mouse embryo tissue lysate
Lane 8 : Mouse lung tissue lysate
Lane 9 : Rat lung tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

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.

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.

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.

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.

All lanes : 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
Lane 2 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa


Exposure time: 48 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Smad4 antibody [EPR22589-112] (ab230815) at 1/1000 dilution

Lane 1 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

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

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.