Dephosphorylation of pS218 of RGS18 in human platelets treated with forskolin.

<p>A. Time-course of S216 and S218 phosphorylation and binding to 14-3-3. Washed platelets were incubated without or with 10 µM forskolin for the indicated times. Platelets were lysed and lysates were subjected to GST-14-3-3γ pull-down. Precipitates were analyzed by Western blotting using mouse anti-RGS18 antibody (upper panel, RGS18). Lysate blots were incubated with either rabbit anti-pS216pS218 RGS18 (second panel), rabbit anti pS216 RGS18 (third panel) or rabbit anti-RGS18 (lower panel, total RGS18). Shown is a representative example of four independent experiments. B. Quantitation of results shown in A, top panel. Blots of 4 independent experiments were analyzed by densitometry and relative intensities of precipitated RGS18 versus total RGS18 are presented as means +SEM. C. Quantitation of results shown in A, pS216pS218 and pS216 RGS18 panels. Band intensities were determined by densitometry and ratios of pS216/pS218 RGS18 or pS216RGS18 to total RGS18 were calculated. Values were normalized to highest values and presented as means +SEM. D. Effect of phosphatase inhibition on S216/S218 phosphorylation. Washed platelets were incubated without or with 10 µM forskolin or 1 µM okadaic acid as indicated. Platelets were lysed and lysates were subjected to SDS-PAGE and Western blotting. Blots were incubated with either rabbit anti-pS216pS218 RGS18 (upper panel) or rabbit anti-RGS18 (lower panel).