Effects of PKC, PKA and PI3 kinase inhibitors on ET-1-induced activation of ERK1/2 in HASMCs. Serum-starved cells were treated with 500 nM of staurosporin, 10 μM of GF109203X, 5 μM of rottlerin, 10 μM of H-89, or 2 μM of wortmannin for 30 min prior to addition of ET-1. A, bar graph shows inhibitory effects of inhibitors on phosphorylated ERK1/2 induced by 10 nM of ET-1. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. B, inhibitory effects of inhibitors on phosphorylated ERK1/2 activity induced by 10 nM of ET-1. Phosphorylated ERK1/2 activity was determined by phosphoELISA assay as described in Methods. The upper panel of A indicates representative images of immunofluorescence showing the phosphorylated ERK1/2 from samples treated with inhibitors prior to addition of ET-1. The scale bar in each image represents 20 μm. Data represent mean ± S.E.M. * p < 0.05, **p < 0.01, *** p < 0.001 compared with the ET-1-stimulated states after DMSO treatment. p = phosphorylation.