Gtx70214

Coverslips were placed in the 6-well plates of cultured cells, collected when the cells grew to 70 to 90%. The coverslips were fixed by 4% paraformaldehyde (Servicebio) for 15 minutes, covered, and blocked with 3% bovine serum albumin (BSA; Servicebio) for 30 minutes at room temperature (if the primary antibody was from goat, 10% normal rabbit serum was used to block it), and then incubated with the primary antibody (ZO-1, Servicebio, GB111402, 1:800; Na⁺/K⁺-ATPase, Santa, sc-21712, 1:50; Bax, Abcam, ab32503, 1:200; Bcl-2, Abcam, ab182858, 1:200; caspase-3, Abcam, ab13847, 1:200; caspase-9, Abcam, ab202068, 1:200; NFκB p65, Servicebio, GB11142, 1:100; NLRP3, Servicebio, GB11300, 1:500; ASC, Proteintech Group, 40500-1-AP, 1:100; caspase-1, Boorsen, BS-10743R, 1:200; IL-1β, Servicebio, GB11113, 1:200; FoxO3a, CST, 2497, 1:200; p53, Gene Tex, GTX70214, 1:500; p21, Gene Tex, GTX629543, 1:200) overnight in a wet box at 4°C. The cell samples were covered with secondary antibodies of the corresponding species (Cy3 labeled goat anti-rabbit, Servicebio, GB21303, 1:300; Cy3 labeled goat anti-mouse, Servicebio, GB21301, 1:300), and incubated at room temperature for 50 minutes. The 4',6-diamidino-2-phenylindole (DAPI; ServiceBio) was used to re-stain the nucleus and anti-fade mounting medium (Servicebio) was used to seal the coverslips.