Stimulation of fibrogenic signalling in Col-GFP cells.

<p>(<b>A</b>) Cells were stimulated with indicated concentrations of TGF-β1 (0.1 ng/ml; 1.0 ng/ml), BMP-2 (25 ng/ml), PDGF-BB (25 ng/ml) or left untreated (Co.) for indicated time intervals from 10 min to 4 hrs. When indicated, the cells were treated with the ALK5 inhibitor SB431542 (5 µM). Cell extracts were prepared and cellular proteins tested for expression of PDGFRβ, phosphorylated Smad1/5/8 (pSmad1/5/8) and Smad2 (pSmad2) (I), the phosphorylated forms of p38 (p-p38) and ERK1/2 (pERK1/2), phosphorylated ATF-2 (pATF-2) (II), Id2 and GFP by Western blot. (<b>B</b>) Cells were stimulated for the indicated time intervals with EGF (50 ng/ml; 100 ng/ml). Thereafter, cell extracts were prepared and analyzed by Western blot using specific antibodies to linker phosphorylated Smad2 (pSmad2L), phosphorylated Smad1/5/8 (pSmad1/5/8) (I), the phosphorylated forms of p38 (p-p38) and ERK1/2 (pERK1/2), phosphorylated ATF-2 (pATF-2) (II) and GFP. Membranes (<b>A, B</b>) were incubated with an antibody specific for β-actin to demonstrate equal protein loading. Band intensities of the indicated proteins (<b>A, C</b>) were measured, normalized to β-actin and represented as fold induction of the unstimulated sample. Results show representative images of one of three independent experiments.