Fig 3. ERK1/2 mediates the effect of H-Ras on ENaC.

A, I_{ami (normalized)} in FRT cells co-transfected with all three wild-type α-, β- and γ-ENaC subunits (ENaC) together with pcDNA3.1, scrambled siRNA, an siRNA directed against c-Raf (siRNA c-Raf), H-Ras^G12V or siRNA c-Raf and H-Ras^G12V. B, I_{ami (normalized)}, in FRT cells co-transfected with all three wild-type α-, β- and γ-ENaC subunits and with or without H-Ras^G12V, as indicated. Eighteen hours before experimentation monolayers were treated with 20 μM of a MEK1/2 inhibitor, PD98059, (+) or vehicle (−). In a set of experiments, cells were pre-treated in EGF (100 ng/ml) as indicated. C, Immunoblot analysis of total ERK1/2 or ERK1/2 phosphorylation (ERK1/2-P) in FRT cells transfected with empty pcDNA3.1, H-Ras^G12V, K-Ras^G12V or a constitutively-active mutant N-Ras (N-Ras^G12V). D, Immunoblot analysis of total ERK1/2, ERK1/2-P and H-Ras in FRT cells transfected with empty pcDNA3.1, wt-H-Ras, H-Ras^G12V or H-Ras^S17N. E, Immunoblot analysis of total ERK1/2 and ERK1/2-P FRT cells transfected with empty pcDNA3.1, H-Ras^G12V treated with PD98059 (+) or vehicle (−). F, I_{ami (normalized)} of FRT cells transfected with ENaC together with (+) or without (−) H-Ras^G12V. Cells were pre-treated for 18 hr with SB239063 (p38 inhibitor, 5 μM), SP600125 (JNK inhibitor, 20 μM) or U73122 (PLC inhibitor, 10 μM) as indicated. *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively.