Co-immunoprecipitation assay

After MAECs were lysed on ice with protein lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, with the addition of a protease inhibitor cocktail), they were centrifuged at 12 000× g and 4°C for 20 min. Orai1–3 or VE-cadherin proteins were immunoprecipitated by incubating 800 μg of extracted protein with 5 µg of anti-Orai1–3 antibodies (ProteinTech Group, Chicago, Illinois, USA) or anti-VE-cadherin antibody (Affinity Biosciences, Ohio, USA), respectively, on a rocking platform overnight at 4℃. Protein A agarose was then added and incubated for an additional 3 hours at 4℃. The immunoprecipitates were washed with cell lysis solution, and cell lysates (100 µL) and loading buffer (25 µL) were added. Next, the sample was boiled at 100°C for 10 min. The resulting obtained supernatant was used for protein electrophoresis. For the immunoblots, all of the samples were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using 15% agarose gels, transferred to polyvinylidene fluoride membranes, and probed with the indicated primary antibodies at a dilution of 1:200 in a phosphate-buffered saline (PBS; in mM: 137 NaCl, 2.7 KCl, 10 Na₂HPO₄, 1.8 KH₂PO₄, pH 7.4) containing 0.1% Tween-20 and 5% non-fat dry milk. Immunodetection was accomplished using horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Affinity Biosciences) followed by an enhanced chemiluminescence plus detection system (Peiqing, Shanghai, China).