Figure 2. Effects of BRCA1 on Smad3 protein.

(A) Wild type BRCA1 and Smad3 cooperate to increase p3TP-Lux promoter activity. 0.1 µg of p3TP-Lux and 10 ng of pCMVβ-galactosidase plasmids were co-transfected with 10 ng of Flag-Smad3, 50 ng of HA-Smad4, and 0.1 µg of wild type HA-BRCA1 plasmids into COS-7 cells, as indicated. 36 hrs after transfection, luciferase and β-galactosidase assays were performed. The luciferase activities were normalized to β-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. *, P<0.05 versus empty vector only. (B) Wild type BRCA1 increases Smad3 protein abundance. 0.15 µg of Flag-Smad3 plasmid was co-transfected with 0.1 µg, 0.25 µg, and 0.5 µg of wild type HA-BRCA1 or 0.1 µg and 0.25 µg of caTβRII into HEK293 cells. 36 hrs after transfection, 10 µg of total cell lysates was subjected to Western blotting with anti-Flag monoclonal antibody. Anti-CSK polyclonal antibody was used to monitor equal loading. Flag-Smad3 expression levels were normalized to CSK and are represented graphically. This is a representative experiment out of 4 experiments. (C) BRCA1 mutants could not increase Smad3 protein abundance. 0.15 µg of Flag-Smad3 was co-transfected with 0.1 µg of wild type or various mutant forms of HA-BRCA1 into HEK293 cells. 10 µg of total cell lysates was subjected to Western blotting with anti-Flag monoclonal antibody. Anti-CSK polyclonal antibody was used to monitor equal loading. Flag-Smad3 expression levels were normalized to CSK and are represented graphically. This is a representative experiment out of 4 experiments.