FIGURE 3.
Activation of Pyk2 downstream of G12/13 and outside-in signaling. A, washed human platelets were stimulated in the presence of 100 nm YM254890 with 500 μm AYPGKF for various time points and probed with anti-phospho-Pyk2 (Tyr-402 and Tyr-881), anti-phospho-Akt (Ser-473), or anti-β-actin (lane loading control) antibodies by Western blotting. B, platelets were stimulated with 100 nm 2-MeSADP for 2 min in the presence and absence of 100 μm MRS2179, 100 nm AR-C69931MX, or 10 μm SC57101 and probed with anti-phospho-Pyk2 (Tyr-402), anti-phospho-Akt (Ser-473), or anti-β-actin (lane loading control) antibodies by Western blotting. C, densitometric measurement of phospho-Pyk2 and phospho-Akt, expressed as fold-increase over control. Data are mean ± S.E. (n = 3). *, p < 0.005 compared with agonist. D, lysates from non-adherent (BSA) and fibrinogen-adherent (Fib) platelets were probed with anti-phospho-Pyk2 (Tyr-402 and Tyr-881), anti-phospho-Src (Tyr-416), or anti-β-actin (lane loading control) antibodies. The blot shown is representative of three independent experiments.