Fig. 6.
Fig. 6. Wortmannin does not inhibit GPIb-stimulated Src association with GPIb, tyrosine phosphorylation of FcRγ chain, Syk, or PLCγ2. / (A-B) Platelets were preincubated with vehicle solution as the control or 100 nM wortmannin (WM) at 37°C for 10 minutes, and then activated with 6 μg/mL botrocetin plus 10 μg/mL VWF for the indicated time periods (seconds). GPIb was immunoprecipitated from lysates and analyzed by immunoblotting with anti-Src and anti-GPIb MoAbs (A). The cell lysates were precipitated with GST-Syk-SH2, anti-Syk, or anti-PLCγ2 antibodies, respectively. The precipitated proteins were resolved on SDS-PAGE. The membranes were immunoblotted for phosphotyrosine using 4G10 plus PY20 (B; pY). Then the membranes were stripped and reprobed for Syk and PLCγ2 using corresponding antibodies (B, lower panels).

Wortmannin does not inhibit GPIb-stimulated Src association with GPIb, tyrosine phosphorylation of FcRγ chain, Syk, or PLCγ2.

(A-B) Platelets were preincubated with vehicle solution as the control or 100 nM wortmannin (WM) at 37°C for 10 minutes, and then activated with 6 μg/mL botrocetin plus 10 μg/mL VWF for the indicated time periods (seconds). GPIb was immunoprecipitated from lysates and analyzed by immunoblotting with anti-Src and anti-GPIb MoAbs (A). The cell lysates were precipitated with GST-Syk-SH2, anti-Syk, or anti-PLCγ2 antibodies, respectively. The precipitated proteins were resolved on SDS-PAGE. The membranes were immunoblotted for phosphotyrosine using 4G10 plus PY20 (B; pY). Then the membranes were stripped and reprobed for Syk and PLCγ2 using corresponding antibodies (B, lower panels).

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