Fig. 8. Effect of expressing a Y397F FAK mutant on TGF1-induced Smad3 phosphorylation. (A) Wild type (WT) or Y397F FAK was transfected along with a vector expressing Flag-tagged Smad3 in HKCs for 20 hours, followed by treatment with TGF1 (1.0 ng/ml) or vehicle for 30 minutes. Expressed Smad3 was immunoprecipitated with anti-Flag affinity gel and Smad phosphorylation was evaluated with phospho-specific antibodies. LB, lysis buffer control for immunoprecipitation. (Bottom) Intensities of the immunoreactive bands for phosphorylated Smad3 at either the C-terminus [423/425; pSmad3(C)] or linker region [207/212; pSmad3(L)] were corrected for expression levels of Smad3 construct detected as Flag expression and demonstrated graphically (mean ± s.e.m., n=6, *P<0.05 compared to those with wild-type FAK with TGF1). (B) Flag-tagged Smad3 wild-type or His-tagged Smad3 EPSM constructs were transfected. Expressed proteins were immunoprecipitated with appropriate anti-tag antibody and Smad3 phosphorylation levels either for linker-region (top panel) or C-terminal (second panel) serines were evaluated. Expression of the Smad3 constructs detected by the corresponding antibody to the tag is also shown (bottom panels).