Cyclic nucleotide signaling removes 14-3-3 from RGS18 in platelets. (A-C) Washed platelets were incubated without or with forskolin (10μM, 30 minutes) or SNP (10μM, 10 minutes), followed by the addition of thrombin (0.1 U/mL, 30 seconds; A), thromboxane mimetic U46619 (1μM, 1 minute (B), or ADP (10μM, 1 minute (C). Platelets were lysed and lysates were subjected to GST-14-3-3γ pull-down. After SDS-PAGE and blotting pull-down assays, samples were incubated with mouse anti-RGS18 Ab (top panel, RGS18), and lysates were either incubated with rabbit anti-RGS18 Ab (bottom panel, total RGS18) or rabbit anti–pS7-Rap1GAP2 Ab, which was shown in Figure 1A and B to recognize phosphorylated S216 of RGS18 (middle panel, pS216 RGS18). Shown are examples of experiments performed at least 3 times. (D-F) Blots of 3-5 independent experiments shown in panels A through C were analyzed by densitometry, and data are expressed as means ± SEM. (D) Densitometry of the thrombin experiment (panel A). (E) Thromboxane mimetic U46619 (panel B). (F) ADP data (panel C). Statistical significance of relative 14-3-3 binding was detected using 1-way ANOVA in combination with the Tukey posttest, as indicated. ****P < .0001; ***P < .001; **P < .01; *P < .05.