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Fig. 5. Overexpressed caMEK restores phosphorylation of the Smad3 linker region and–0.4
2(I) collagen promoter activity in cells cultured on pLL. (A) HKCs were transfected with a construct that expresses caMEK, along with an Elk-Gal transactivation system (left-hand graph) or the–0.42(I) collagen promoter (right-hand graph) and then the cells were re-plated on either gelatin-or pLL-coated six-well plates. TGF
1-induced reporter activity was corrected for -galactosidase expression and representative results (mean ± s.e.m.) performed in triplicate from one of three experiments are shown as a graph. Numbers in the graph indicate relative increase of Gal4-Elk-luc activity over control in the absence of TGF1 (left-hand graph) or fold induction of 2(I) collagen promoter activity by TGF1 over each control (right-hand graph). White bars, vehicle; black bars, TGF1 (1.0 ng/ml, 12 hours). (B) HKCs transfected with constructs expressing Flag-Smad3 and caMEK were re-plated on either gelatin or pLL and cultured for another 3 hours followed by TGF1 treatment (1.0 ng/ml, 30 minutes). Expressed Smad3 was immunoprecipitated with anti-Flag affinity gel and phosphorylation was evaluated with phospho-specific antibodies (top panels). Y397-FAK phosphorylation and Flag expression using whole-cell lysate (WCL) are shown in the bottom panels. LB, lysis buffer control for immunoprecipitation.
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