Fig. 5. Agonist-induced changes in phosphorylation of MLC, CPI-17 and MYPT1 in cultured VSMCs. (A) MLC phosphorylation is expressed as percent of total MLC (see Materials and Methods). Either ATII (1.0 µM) or ET-1 (0.1 µM) was added at 0 minutes. Inset shows representative 2D gel patterns of 20 kDa MLC of cultured VSMCs at rest (control), stimulated with ATII (+ATII) and ET-1 for 2.5 minutes (+ET-1). (B-D) Western blot results. Of each protein extract 20 µg were loaded onto two identical polyacrylamide gels and each was transferred onto nitrocellulose membrane. The total amount of (nonphosphorylated and phosphorylated) MYPT1 or CPI-17 was then determined with respective antibodies on one membrane (see Materials and Methods). We then compared the ratios of phosphorylated CPI-17 or MYPT1 to the total CPI-17or MYPT1 in the paired set of western blots and expressed relative phosphorylation levels as the percentage of control phosphorylation. (B1,B2) Western blots of total (CPI-17) and phosphorylated CPI-17 (pCPI-17) in cultured VSMCs at rest or VSMCs stimulated for 1, 2.5 and 5 minutes with ET-1 and for 5 minutes with the activator of PKC PDBu (B1). For average CPI-17 phosphorylation, the response to the 5-minute incubation with PDBu was normalized to 1 (B2). (C,D) Phosphorylation of MYPT1 at (C) Thr696 and (D) Thr853 after 2.5-minute incubation with ET-1 (0.1 µM) or ATII (1.0 µM). Responses in the presence of ET-1 were normalized to 1; ^*, significantly different to control (CT); n=3-5; P0.05.