Bs 20608r

Cells were lysed on ice with RIPA buffer (P0013B, Beyotime, Shanghai, China), and the supernatant was collected after centrifugation. Then, 10% SDS-PAGE gel electrophoresis was carried out, followed by being transferred to a PVDF membrane. After a 2-h block in a western blotting buffer (5% non-fat milk), the membrane was incubated overnight in a shaker with primary antibody dilution. The primary antibodies used specifically were OPN (bs-23258R, rabbit, 1:1000, Bioss, Beijing, China; ab63856, rabbit, 1:1000, Abcam, Cambridge, UK), Orai1 (ab111960, rabbit, 1:1000, Abcam, Cambridge, UK; 28411-1-AP, rabbit, 1:500, Proteintech, Wuhan, China), STIM1 (11565-1-AP, rabbit, 1:1000, Proteintech, Wuhan, China), eNOS (bs-20608R, rabbit, 1:1000, Bioss, Beijing, China), p-eNOS (bs-3589R, rabbit, 1:1000, Bioss, Beijing, China), and GAPDH (TA-08, mouse, 1:2000, Zsbio, Beijing, China). Afterward, HRP-conjugated secondary antibodies were diluted in a secondary antibody dilution solution, specifically goat anti-rabbit (ZB-2301, Zsbio, Beijing, China) and goat anti-mouse (ZB-2305, Zsbio, Beijing, China) at a ratio of 1:20000. After 2 h of incubation at room temperature, an ECL detection kit (340958, Thermo, Massachusetts, USA) was used to detect the proteins. The density quantification of the immunoblot was carried out with a chemiluminescent gel imaging analysis system (Bio-Rad, California, USA).