TPO-induced intracellular signaling depends on intracellular and extracellular calcium. (A) Western blot analysis of STAT5, AKT, and ERK1/2 phosphorylation (p-STAT5, p-AKT and p-ERK1/2 respectively) after 10 minutes stimulation with recombinant human thrombopoietin (rhTPO) (100 ng/mL) in the presence or not of Ethylene Glycol Tetraacetic Acid (EGTA). Total STAT5, AKT and ERK1/2 were stained to ensure equal loading. Band densitometry analysis derived from 3 independent experiments is shown (P < .05). (B) Western blot analysis of p-STAT5, p-AKT, and p-ERK1/2 after 10 minutes of stimulation with rhTPO (100 ng/mL) in the presence or not of BAPTA-AM. Total STAT5, AKT, and ERK1/2 were stained to ensure equal loading. Band densitometry analysis derived from 3 independent experiments is shown (P < .05). (C) In physiological calcium (Ca²⁺) concentration (1.5 mM), stimulation with rhTPO (100 ng/mL) evokes an initial rise in [Ca²⁺]_i followed by a plateau, which is further increased by concomitant stimulation with CPA. The statistical analysis of peak fluorescence intensities is shown by box plot depicting the upper and lower values (lowest and highest horizontal line, respectively), lower and upper quartile with median value (box), and outside values (dots). Overall, data derive from 82 measurements from 3 independent experiments (P < .05). (D) Western blot analysis of p-STAT5, p-AKT, and p-ERK1/2 after 60, 600, 1200, 1800, or 3600 seconds of stimulation with rhTPO (100 ng/mL) in the presence or not of CPA. Total STAT5, AKT, and ERK1/2 were stained to ensure equal loading.