Time course of JAK2 and Src signaling triggered by 20 mU/mL thrombin. (A-E) Normal (1-5) or ET platelets (6-10) were challenged with 20 mU/mL thrombin for the indicated time in the presence of vehicle (lanes 1-4, 6-9), or 10μM PP2 (lanes 5,10). (A) Platelets were lysed in IP-buffer and immunoprecipitated with anti-JAK2 antibody and the IPs were subjected to SDS/PAGE, blotted and immunostained with anti–P-JAK2 antibody, stripped, and reprobed with anti-JAK2 antibody. (B) Platelet lysates were immunoprecipitated with anti-Src antibody and the IPs were immunostained with the phospho-specific anti–Src-P-Tyr416 antibody, and reprobed with anti-Src antibody. (C) Platelet lysates were immunoprecipitated with anticortactin antibody and the IPs were immunostained with anti–P-Tyr antibody, and reprobed with anticortactin antibody. (D) IPs obtained by treatment with anti-STAT5 antibody were immunostained with anti–P-STAT5 antibody, and reprobed with anti-STAT5 antibody. (E) IPs obtained with anti-ERK1/2 antibody were immunostained with anti–P-ERK1/2 antibodies, and reprobed with anti-ERK1/2 antibody. Means of densitometric values are reported above the relative phosphorylated bands. Panels A, C, and D are representative of experiments performed with 12 normal and 16 ET samples either JAK2^V617F negative or positive, whereas panels B and E are representative of experiments performed with 6 normal and 6 ET samples either JAK2 V617F negative or positive. **P < .001 versus the relative protein Tyr phosphorylation detected after 2 minutes of thrombin stimulation in the absence of PP2.