Time course and concentration-effects of ET-1 on activation of ERK1/2. Cultured HASMCs were starved for 24 h in serum-free medium and then stimulated with ET-1. A, bar graph shows time-dependent activation of ERK1/2 by ET-1 at 1 μM. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. B, representative autoradiograph of western blot showing the level of phosphorylated and total ERK1/2 from the samples treated with ET-1 at 0 min, 5 min,10 min and 15 min. C, Bar graph shows concentration-dependent activation of ERK1/2 by ET-1 from 1 nM to 1 μM for 10 min. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. The upper panels of A and C indicate representative images of immunofluorescence showing the phosphorylated ERK1/2 from the samples treated with ET-1 at different time points and various concentrations, respectively. The scale bar in each image represents 20 μm. Data represent mean ± S.E.M. *** p < 0.001 compared with the vehicle value. p = phosphorylated; t = total.