FIG. 5.

Regulation of ERK phosphorylation by Rho family proteins. (A) Myocytes were transfected with ERK^Myc and inhibitory mutants of Rho family proteins. Following exposure to ET-1 (100 nM, 5 min), ERK^Myc was immunoprecipitated and immunoblotted with antibodies to phosphorylated ERK (upper panel). Parallel blots were stripped and probed with an antibody to the c-Myc tag (lower panel). The experiment was repeated on three separate occasions with similar results. (B) Myocytes were transfected with ERK^Myc, ΔN-Raf, and/or activated mutants of Rho family proteins. When applicable, exposure to ET-1 (100 nM) was for 5 min. ERK^Myc was immunoprecipitated and immunoblotted with antibodies to phosphorylated ERK (upper panel). Parallel blots were reprobed with an antibody to the c-Myc tag (lower panel). The experiment was repeated on four separate occasions with similar results. (C) Blots shown in panel B were analyzed by scanning densitometry, and the amount of phospho-ERK^Myc was adjusted for total ERK^Myc protein. Results are means ± SEM for four separate myocyte preparations. ∗, P < 0.05 relative to ΔN-Raf alone (unpaired two-tailed t test).