Fig. 4. Effects of pLL on Smad and ERK signals. (A) Cells on either gelatin-or pLL-coated plates were prepared as described in Fig. 3 and total protein harvested after TGF1 treatment (1.0 ng/ml, 30 minutes) was subjected to immunoblotting with anti-linker-region phospho-Smad3 [207/212; pSmad3(L)], anti-C-terminal phospho-Smad3 [423/425; pSmad3(C)] and anti-Smad2/3 antibodies. Representative blots from one of five independent experiments are shown. WCL, whole-cell lysate. (Right) Intensity of the immunoreactive bands for C-terminal (triangles; white, gelatin; black, pLL) or linker-region (squares) phosphorylation of Smad3 were corrected for Smad3 expression levels (y axis) and plotted against phospho-Y397 FAK levels corrected for FAK expression (x axis) in each corresponding experiment (representative blots for FAK shown in Fig. 2C). (B) Cells were transiently transfected with a plasmid that expresses an activation domain of Elk linked to the DNA-binding region of yeast Gal4, along with a Gal4-luciferase reporter construct, then replated on either gelatin or pLL. After 20 hours of TGF1 treatment, cells were harvested and luciferase activity was analyzed to indicate Elk activation. Graph shows mean ± s.e.m. (n=3) of luciferase activity after correction for -galactosidase expression from five separate experiments. Numbers in the graph represent fold induction by TGF1 over control. White bars, vehicle; black bars, TGF1 (1.0 ng/ml 20 hours). *P<0.05, compared to values on gelatin without TGF1.