Usp24

Cells harvested from 10-cm dishes were lysed in RIPA buffer on ice for 30 min. The supernatant was collected after centrifugation (12000 g, 20 minutes). Add 4X loading buffer added into the supernatant and boiled in hot water (100 °C) for 10 min. The supernatant was then subjected to electrophoresis on SDS-PAGE gels. Sealed the protein-containing PVDF membrane with 5% skim milk for 1 hour, incubate this membrane with primary antibody at 4 °C overnight. Wash the membrane with TBST buffer (3 times, 5 minutes each time) and incubate the membrane with secondary antibody for 2 hours at room temperature. Chemiluminescence Western Blot Detection Kit (Cat. No. 32209, Thermo Fisher Scientific, USA) was used to measure the chemiluminescence. Primary antibodies used for Western blotting: USP24 (Proteintech, 13126-1-AP; 1:1000 dilution), GSDMB (Proteintech, 12885-1-AP; 1:1000 dilution), GAPDH (Abcam, ab8245, 1:5000 dilution), STAT3 (Cell Signaling Technology, 30835S; 1:2000 dilution), p-STAT3 Y705 (Cell Signaling Technology, 9145S; 1:1000 dilution), and HA tag antibody (Abcam, ab18181, 1:5000 dilution). ImageJ software (National Institutes of Health) was used to evaluate protein levels.

The following antibodies were used: beta‐actin (1:10,000, mouse), BIRC8 (mouse, Abnova # H00112401‐B01), cIAP1/2, cleaved caspase‐3‐5A1E (1:300, rabbit, Cell Signaling #9664), CUL1 (1:500, mouse, Invitrogen #32‐2400), cyclin A (1:1,000, mouse, Santa Cruz #sc‐751), cyclin B1 (1:1,000, mouse, Cell Signaling #4138), cyclin E (1:1,000, mouse, kind gift of M. Pagano), FLAG (1:1,000, rabbit, Sigma #F7425), FLAG‐M2 (1:1,000, mouse, Sigma #F3165), HA‐16B12 (1:2,000, mouse, Covance #MMS‐101P), human MCL1 (1:500, rabbit, BD Pharmingen #554103), murine MCL1 (1:1,000, rabbit, A&D Serotec AHP1249), ML‐IAP (mouse, Santa Cruz #sc‐71592), NIAP (rabbit, Abcam #ab25968), PARP1/2 (1:1,000, rabbit, Santa Cruz #sc‐7150), pHH3 (S10) (1:300, rabbit, Cell Signaling #9701), PLK1 (1:500, rabbit, Invitrogen #33‐1700), survivin (1:3,000, rabbit, R&D Systems #AF886), USP7 (rabbit, Bethyl Lab. #A300‐033A), USP9X (1:4,000, rabbit, Bethyl Laboratories #A301‐351A), USP10 (rabbit, Bethyl Lab. #A300‐900A), USP24 (Proteintech Europe, #13126‐1‐AP), USP28 (rabbit, Bethyl Lab. #A300‐898A), V5 (1:1,000, rabbit, Sigma #V8137), XIAP (1:1,000, mouse, BD Biosciences #610716), XIAP (1:1,000, rabbit, Cell Signaling #2042), and XIAP (1:1,000, R&D Systems #AF8221).