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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: Exp Cell Res. 2013 Sep 27;319(19):2928–2937. doi: 10.1016/j.yexcr.2013.07.013

Figure 4. Effects of MEK/ERK inhibition on Smad3LR phosphorylation and Smad3-mediated COL1A2 promoter activity in renal epithelial cells.

Figure 4

A, S204 phosphoacceptor is not an ERK target in renal epithelial cells. Renal epithelial cells (HKC) were pre-treated (1 h) with vehicle (DMSO) or PD0325901 (5 nM) prior to vehicle- or TGF-β1 (2 ng/ml) stimulation (1 h). Phosphorylations at T179, S204, S208, S213 and S423/S425 were analyzed by western blotting with specific phospho-peptide Smad3 antibodies. S204 phosphorylation is not prevented by the MEK/ERK inhibitor. B, Smad3-mediated COL1A2 promoter activity in renal epithelial cells does not require ERK. HKC were transfected with β-galactosidase, COL1A2-luciferase (upper panel) or Elk-gal (lower panel) and constructs for 3 hours. The pFA-Elk plasmid expresses an activation domain of Elk linked to the DNA-binding region of yeast Gal4, along with a Gal4-luciferase reporter construct. Transfected cells were then pre-treated with vehicle (DMSO) or PD98059 (20 μM) for 1 h, before vehicle- or TGF-β (2 ng/ml) stimulation for a further 20 h. Relative luciferase activity (mean ± S.E.M., n=3) corrected for β-galactosidase activity. Shown here is a representative experiment of three independent experiments. *P<0.05, **P<0.01 vs. vehicle-treated cells. δδδP<0.001 vs. TGF-β-treated cells.