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. 2015 Oct 27;290(50):29974–29983. doi: 10.1074/jbc.M115.678003

FIGURE 1.

FIGURE 1.

Cyclic nucleotide signaling inhibits thrombin and collagen induced Rac1 activation. A, effects of PGE1 and SNP on thrombin-induced Rac1 activation. Washed human platelets were pre-incubated without or with PGE1 (0.5 μm, 1 min) or SNP (10 μm, 10 min) followed by thrombin (0.1 unit/ml, 1 min). Platelets were lysed, and pull-down assays were performed using GST-PAKI-PBD followed by Western blotting. Quantitative data on Rac1-GTP (upper panel) and total Rac1 (lower panel) levels were obtained using an Odyssey Classic scanner. Below the panels the ratios of active versus total Rac1 normalized to the untreated controls are shown for three independent experiments (n1–3). B, effects of cAMP and cGMP analogues on thrombin-induced Rac1 activation. Platelets were incubated with Sp-5,6-DCl-cBIMPS (cAMP analogue, 300 μm, 30 min) or 8-pCPT-cGMP (cGMP analogue, 300 μm, 30 min) followed by thrombin (0.1 units/ml, 30 s). Rac1-GTP and total Rac1 levels were determined as described in A, and Rac1-GTP/total Rac1 ratios are shown below the panels. C, effects of PGE1 and SNP on collagen-induced Rac1 activation. Platelets were incubated with PGE1 (0.5 μm, 1 min) or with SNP (10 μm, 10 min) followed by collagen (95 μg/ml, 1 min). Rac1 levels were analyzed as described in A.