Figure 4. Effects of H₂O₂ on the association and colocalization between Smad3 and BRCA1.

(A & B) H₂O₂ increases Smad3 and BRCA1 interaction in HaCaT and HCCBRCA1 cells. Cells were treated with TGF-β1 (2 ng/ml) and H₂O₂ (100 µM) for 1 hour, as indicated. Total cell lysates were subjected to immunoprecipitation with anti-Smad3 polyclonal antibody and Western blotting with anti-BRCA1 polyclonal antibody. The increases in Smad3-binding to BRCA1 were normalized to Smad3 protein expression and are represented graphically. The expression levels of Smad3 and BRCA1 proteins were determined in 20 and 30 µg of total cell lysates, respectively. The shifted bands of BRCA1 after H₂O₂ treatment are shown in HaCaT cells. This is a representative experiment out of 4 experiments. (C) H₂O₂ increases phospho-Smad3 and BRCA1 colocalization in HCCBRCA1 cells. HCCBRCA1 cells were treated with 2 ng/ml of TGF-β1 and 100 µM of H₂O₂ for 1 hour as indicated. Anti-BRCA1 monoclonal antibody (Ab-3) and anti-phospho-Smad3 polyclonal antibody were used to immunostain BRCA1 protein (Red) and phospho-Smad3 protein (Green), respectively. Merge shows the colocalization of BRCA1 and phospho-Smad3 protein. This is a representative experiment out of 4 experiments.