(A) Tyrosine phosphorylation of STAT3. Platelets were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100 before or 5 minutes after stimulation with hTPO (100 ng/mL) or hG-CSF for 5 minutes. STAT3 was immunoprecipitated from 6.0 × 108 platelets from littermate mice or 2.0 × 108 platelets from Tg mice. Immune complexes were resuspended in SDS-sample buffer and divided into two. Proteins were separated by 15 to 7.5% SDS-PAGE and transferred onto PVDF membranes. One immunoblot was probed with anti-phosphotyrosine antibodies and bands were visualized by chemiluminescence (top). The other blot was probed for STAT3 (bottom). The arrows indicate the bands of interest. (B) Tyrosine phosphorylation of STAT5. The same as in (A) except the combination of STAT5A and B antisera were used instead of STAT3 antisera. (C) Tyrosine phosphorylation of Jak2. The same as in (B) except the combination of an anti-Jak2 polyclonal antibody was used instead of STAT5 antisera.
