Activation of JAK/STAT signaling pathways in MPN platelets. (A) Total platelet lysates from MPN patients (JAK2-WT and V617F) and healthy donors (N) were stimulated (+) or not (−) for 10 minutes with TPO (100 ng/mL) and then subjected to immunoblot analysis with anti-phospho-JAK2 (p-JAK2) and anti-phospho-STAT5 (p-STAT5) antibodies. Total JAK2 and STAT5 levels were analyzed with anti-JAK2 and anti-STAT5 antibodies. Blot quantification was performed with multi Gauge software (Fujifilm). Numbers under the phospho-JAK2 blots show the ratios (arbitrary units) of phospho-JAK2/total JAK2. Actin blot is shown as loading control; numbers under this blot denote the mRNA expression level of LNK for each patient. (B) Platelet lysates from healthy donors (N), JAK2-WT and V617F MPN patients were stimulated with or without TPO for 10 minutes and immunoprecipitated with anti-JAK2 antibodies. Western blot analysis with anti-phosphotyrosine (anti-p-Tyr) antibodies allowed identification of phosphorylated JAK2 protein (top panel). Total JAK2 protein expression is shown (bottom panel). Numbers under the p-Tyr blots show the ratios (arbitrary units) of phospho-JAK2/total JAK2. ET, essential thrombocythemia; PV, polycythemia vera; n.d., not done. (C) Graphic representation of JAK2-fold activation. Data represent the normalized ratios of TPO-induced phospho-JAK2/nonstimulated phospho-JAK2 from (A). Statistical significance was determined using the Student t test: *P = .0025; **P = .0386. (D) Graphic representation of JAK2 phosphorylation status, data represent the normalized ratios of p-TyrJAK2/total JAK2 (arbitrary units) from panel B, ***P = .047.