Stimulation-induced phosphorylation of RasGRP3. (A) Lysates from unlabeled Ramos B cells were unstimulated or treated with 100 nM PMA or 10 μg/mL anti-IgM. RasGRP3 was immunoprecipitated before incubation with a protein phosphatase in the absence or presence of the phosphatase inhibitor vanadate. The resultant immune complexes were separated by 7.5% SDS-PAGE followed by immunoblotting. (B) Ramos B cells were metabolically labeled for 4 hours with ³²P_i and were either unstimulated or stimulated with 10 μg/mL anti-IgM for the last 10 minutes of incubation. Cells were lysed, and RasGRP3 was immunoprecipitated from postnuclear supernatants. Proteins were separated in 7.5% SDS-PAGE and visualized by autoradiography. (C) Densitometric analysis was used to determine the amount of ³²P_i incorporated. The average value obtained with stimulated cells is shown normalized to the control value (n = 3; error bar represents standard deviation of the mean).