Anti SMAD3  

Western Blot of Rabbit anti-SMAD3 antibody. Marker: Opal Pre-stained ladder (p/n MB-210-0500). Lane 1: HEK293 lysate (p/n W09-000-365). Lane 2: HeLa Lysate (p/n W09-000-364). Lane 3: MCF-7 Lysate (p/n W09-000-360). Lane 4: Jurkat Lysate (p/n W09-000-370). Lane 5: A549 Lysate (p/n W09-001-372). Lane 6: HL-60 Lysate (p/n W09-001-GL3). Lane 7: Raji Lysate (p/n W09-001-368). Lane 8: NIH/3T3 Lysate (p/n W10-000-358). Load: 35 μg per lane. Primary antibody: SMAD3 antibody at 1:5,000 for overnight at 4°C. Secondary antibody: Peroxidase rabbit secondary antibody (p/n 611-103-122) at 1:30,000 for 60 min at RT. Blocking Buffer: 1% Casein-TTBS (p/n MB-082) for 30 min at RT. Predicted/Observed size: 48 kDa for SMAD3.

Western Blot of Rabbit Anti-SMAD3 antibody. Lane 1: AML12 unstimulated. Lane 2: AML12 stimulated with TGFB. Lane 3: MEFwt unstimulated. Lane 4: MEFwt stimulated with TGFB. Lane 5: MEF Smad3 KO unstimulated. Lane 6: MEF Smad3 KO stimulated with TGFB. Lane 7: HEK293 Smad3T179A mutant unstimulated. Lane 8: HEK293 Smad3T179A mutant stimulated with TGFB. Lane 9: HEK293 Smad3T179V mutant unstimulated. Lane 10: HEK293 Smad3T179V mutant stimulated with TGFB. Load: 35 μg per lane. Primary antibody: SMAD 3 antibody at 1:1000 for overnight at 4°C. Secondary antibody: IRDye800？ rabbit secondary antibody at 1:10,000 for 45 min at RT. Block: 5% BLOTTO overnight at 4°C. Predicted/Observed size: 48.1kDa. Other band(s): Smad2pT220.

ELISA results of purified Rabbit anti-SMAD3 Antibody tested against BSA-conjugated peptide of immunizing peptide. Each well was coated in duplicate with 0.1μg of conjugate. The starting dilution of antibody was 5μg/ml and the X-axis represents the Log10 of a 3-fold dilution. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using 3% fish gel, Goat anti-Rabbit IgG Antibody Peroxidase Conjugated (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) (p/n 611-103-122) and TMB ELISA Peroxidase Substrate (p/n TMBE-1000).

Rabbit anti-SMAD antibody follows the canonical TGF-β signaling pathway. TGF-β dimers bind to a receptor thereby activating the pathway. The type I receptor then recruits and phosphorylates a receptor regulated SMAD (R-SMAD) .i.e. SMAD2 or SMAD3. The R- SMAD then binds to the common SMAD (coSMAD) i.e. SMAD4, and forms a heterodimeric complex. This complex then enters the cell nucleus and acts as a transcription factor.