TPO, but not thrombin, activates the JAK2/PI3 kinase pathway. Platelets were stimulated with 100 ng/mL TPO (A), various concentrations of TPO (B), or 0.2 U/mL thrombin (C) and lysed in 4× NuPage lysis buffer at the indicated time interval (A,C) or after 5 minutes (B). Alternatively, platelets were stimulated with 100 ng/mL TPO or 0.2 U/mL thrombin for 2 or 10 minutes and extracted in the indicated extraction buffer (RIPA or NP40) followed by immunoprecipitation of JAK2 and STAT5 (D). Lastly, platelets were incubated with the indicated concentrations of JAK2i IV for 15 minutes followed by stimulation with 100 ng/mL TPO (E) or 0.2 U/mL thrombin (F-G) for 5 minutes and lysis in 4× NuPage sample buffer. Platelet lysates and JAK2/STAT5 immunoprecipitates were immunoblotted with the indicated antibodies. Results shown are representative of at least 3 independent experiments.