Effect of TPO on Src and JAK2 activation in normal and ET platelets. (A) Platelets from healthy donors (lanes 1-5) or ET patients (lanes 6-10) were treated with 200 ng/mL TPO (lanes 2-4, 7-9) or 20 mU/mL thrombin (lanes 5,10) for the indicated times. Cells were lysed and aliquots of the lysates were analyzed by Western blot with anti–P-Tyr antibody (top part of the panel). Platelets were lysed in IP-buffer. Aliquots of the lysates were immunoprecipitated with anti-Src antibody and the IPs were immunostained with anti–Src-P-Tyr416 and reprobed with anti-Src antibodies (middle part of the panel). Aliquots of the lysates were immunoprecipitated with anti-JAK2 antibody and the IP were immunostained with anti-P-JAK2 antibody and reprobed with anti-JAK2 antibody (bottom part of the panel). Means of densitometric values relative to the phosphorylated kinases are reported above the relative bands. (B) Platelets from healthy donors (lanes 1-6) or ET patients (lanes 7-12) were treated without (odd lanes) or with (even lanes) 200 ng/mL TPO for 2 minutes in the absence (lanes 1-2, 7-8), or presence of 1μM JAK-inhibitor-I (lanes 3-4, 9-10), or 10μM PP2 (lanes 5-6, 11-12). Platelet lysates were analyzed by Western blot with anti–P-STAT5 antibody and reprobed with anti-STAT5 antibody. Densitometric values of anti–P-STAT5 are reported above the relative bands and are means of data obtained from 6 normal, 3 JAK2^V617-negative, and 3 JAK2^V617-positive ET samples. **P < .001 versus STAT5 phosphorylation in the absence of inhibitors.