Effect of 2-MeSAMP on Rap1 activation in wild-type and Gαi₂^−/− platelets stimulated with ADP and CVX.

(A) Washed wild-type (wt) or Gαi₂-deficient murine platelets were treated with 2-MeSAMP or PBS for 15 minutes, followed by CVX (500 ng/mL, n = 4) or ADP (50 μg/mL, n = 3) for 1 minute. Platelets were lysed and active Rap1 was precipitated and detected as in Figure 1, except a polyclonal antibody was used to detect Rap1. Relative quantities of active Rap1 were determined via densitometry and normalized to levels of active Rap1 in unstimulated platelets on the same blot. Results are plotted as an average fold Rap1 activation ±SEM. (B) In a representative blot the GTP-Rap1 bands in the control lanes are assigned a fold Rap1 activation value of 1.0, indicated by the dashed line in the histogram.