CALR-STIM1 binding is impaired in presence of CALR type I mutation. (A) Top, In situ PLA imaging of STIM1 and CALR interaction (green dots) in CB-derived mature Mks starved (None) or treated with 100 ng/mL rhTPO, 10 μM CPA, or 1 U/mL thrombin (THR). Nuclei are visualized with Hoechst 33258 (blue). Scale bar, 10 μm. Original magnification, ×60/NA1.2. Bottom, Quantification of dot number per single Mk (overall, data derive from 92 measurements; P < .05). (B) CB-derived Mks were treated (+) or not (−) for 10 minutes with rhTPO (100 ng/mL) and lysed. Lysates were immunoprecipitated (IP) with an anti-CALR or anti-STIM1 antibody and subjected to western blotting. Membranes were stained with antibodies against STIM1 and CALR, respectively, and reblotted with the same antibodies to ensure equal immunoprecipitation of the proteins of interest. (C) CB-derived Mks were treated (+) or not (−) for 10 minutes with rhTPO (100 ng/mL) and lysed. Lysates were immunoprecipitated with an anti-ERp57 antibody and subjected to western blotting. Membrane was stained with an antibody against STIM1 and reblotted with anti-ERp57 antibody to ensure equal immunoprecipitation of the protein. β-ACTIN was used as input control.