The role of p38 in agonist- and cGMP-induced phosphorylation of ERK2. (A) 123 cells were transfected with vector (123/vector) or the dominant-negative mutant p38AF cDNA (123/p38AF), 123/vector cells, or 123/p38AF cells were incubated with buffer, ristocetin (1 mg/mL), or ristocetin and VWF (20 μg/mL) at 25°C for 5 minutes. Cells were solubilized by adding equal volume of 2 × SDS sample buffer and immunoblotted with an antibody specific for phosphorylated Thr202/Tyr204 of ERK (New England Biolabs) (P-ERK2) to detect ERK phosphorylation, and with an anti-ERK2 antibody to monitor loading. (B) 123/PKG cells were transfected with vector (PKG/vector) or p38AF cDNA (PKG/p38AF). PKG/vector cells or PKG/p38AF cells were incubated with buffer, ristocetin (1 mg/mL), or ristocetin and VWF (20 μg/mL) at 25°C for 5 minutes and immunoblotted as in panel A. (C) PKG/vector cells or PKG/p38AF cells were incubated with 8-bromo-cGMP (100 μM), 8-pCPT-cGMP (100 μM), or 8-dibutyl-cGMP (100 μM) at 25°C for 5 minutes. Phosphorylation of ERK was analyzed as described in panel A. (D) PKG/vector cells were preincubated with DMSO, SB202190 (20 μM), or SB203580 (20 μM) for 5 minutes and then stimulated with 8-bromo-cGMP (100 μM) at 25°C for 5 minutes. Phosphorylation of ERK was analyzed by Western blot. (E) Washed human platelets were incubated with p38 inhibitor, SB203580, or MEK inhibitor, PD98059, or with DMSO as a control. Platelets were stimulated with botrocetin or botrocetin plus VWF at 37°C for 1 minute, solubilized, and then analyzed for ERK2 phosphorylation as in panel A. (F) Washed human platelets were incubated with p38 inhibitor SB203580 or SB202190, or MEK inhibitor PD98059, or with DMSO as a control. Platelets were stimulated with 0.05 μ/mL thrombin at 37°C for 2 minutes, solubilized, and then immunoblotted to detect ERK phosphorylation by Western blot.