The GPIb-IX–dependent effect of Rac1 inhibitor NSC23766 on thrombin-induced calcium elevation. Control CHO cells and 1b9 cells were loaded with a fluorescent calcium-sensitive dye and then treated with either 0.1% dimethylsulfoxide (DMSO) or 100 μM NSC23766 (NSC). Calcium fluorescence signals of these cells were then recorded following stimulation with (A-B) 3 nM or (C-D) 6 nM thrombin. (A,C) Typical graphs of calcium elevation. (B,D) Quantitative data of A and C (mean ± SEM, 3 experiments; 1-way ANOVA). *P < .05. (E-F) Western blot analysis of levels of GTP-bound active Rac1 in thrombin-stimulated human platelets (E) pretreated with either MPαC or MCsC or in (F) control CHO cells or 1b9 cells. The amount of GTP-bound Rac1 was determined using a PAK-GST pull-down assay, followed by western blotting with an anti-Rac1 antibody. Total levels of Rac1 in platelet lysates were also determined by western blot using anti-Rac1 antibody. (G) Densitometric analysis of western blots as shown in F (mean ± SEM, 3 experiments; **P < .01, 1-way ANOVA) using National Institutes of Health Image J. The results were expressed as arbitrary units of uncalibrated optical density.