TPO-evoked calcium signals depend on SOCE mechanism. (A) Western blot analysis of STIM1 and TRPC1 coimmunoprecipitated with Orai1 after 10 minutes of stimulation with rhTPO (100 ng/mL). Band densitometry analysis of 4 independent experiments is shown (P < .05). (B) Enzyme-linked immunosorbent assay of IP3 formation in Mks after 10 minutes of stimulation with rhTPO (100 ng/mL). Thrombin stimulation (1 U/mL) was used as positive control (n = 7 independent experiments per condition; P < .05). (C) Representative analysis of Ca²⁺ flows in Mks after 10 minutes of stimulation with rhTPO (100 ng/mL), in the presence (blue line) or not (red line) of the IP3 receptor inhibitor 2-APB, in absence of extracellular Ca²⁺ (Ø Ca²⁺) and after addition of physiological extracellular Ca²⁺ concentration (1.5 mM). (D) Western blot analysis of p-STAT5, p-AKT, and p-ERK1/2 after 10 minutes stimulation with rhTPO (100 ng/mL) in the presence or not of the IP3 receptor inhibitor 2-APB. Total STAT5, AKT, and ERK1/2 were stained to ensure the equal loading. The band densitometry analysis of 3 independent experiments is shown (P < .05). (E) Representative analysis of Ca²⁺ flows in Mks after 10 minutes of stimulation with rhTPO (100 ng/mL), in the presence (blue line) or not (red line) of the SOCE inhibitor BTP-2 (20 μM), in physiological extracellular Ca²⁺ concentration (1.5 mM). (F) 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 BTP-2 (20 μM). Total STAT5, AKT, and ERK1/2 were stained to ensure equal loading. The band densitometry analysis of 3 independent experiments is shown (P < .05).