Expression and localization of Cx62 in platelets. (A) The presence of Cx62 was examined by immunoblot analysis of whole-cell lysates from human and mouse whole platelets, MegO1, and HeLa cells using a rabbit polyclonal anti-GJA10 antibody. Actin was used as a loading control. (B-C) The localization of Cx62 in resting and activated (with 5 μM U46619 in the presence of 3 μg/mL integrelin) permeabilized human platelets (0.2% Triton-X-100) was investigated using immunofluorescence microscopy. Cx62 (in red) and membrane GPIb receptors (in green) were stained using anti-GJA10 and anti-GPIb primary antibodies, respectively. Visualization was performed using Alexa-647– and Alexa-488–conjugated secondary antibodies, respectively. (D) The distribution of Cx62 was also studied using super-resolution stochastic optical reconstruction microscopy. Resting and activated permeabilized human platelets were stained using anti-GJA10 and anti-integrin β₃ primary antibodies and visualized using Alexa-647– and Alexa-555–conjugated secondary antibodies, respectively. (E) CBC analysis was performed to determine the levels of Cx62 and β₃ integrin colocalization in resting (0, red line) platelets and after stimulation with thrombin (1 U/mL) for 5 minutes (300, blue line). A CBC value of 0 represents a random distribution, and a positive value indicates closer distribution than expected at random. Data are representative of ≥3 separate experiments.