Sc 56036

5 × 10⁶ cells at the logarithmic growth stage were collected. To extract the total proteins, RAPI lysate (Beyotime, Nanjing, China) containing protease and phosphatase inhibitors was used. The relevant protein concentration was measured using a BCA kit (Thermo Fisher Scientific, MA, USA). After quantification, the proteins were separated using SDS-PAGE. These proteins were transferred to polyvinylidene fluoride (PVDF) membranes and sealed in 5% degrease milk for 1 h. The blots were incubated at 4 ℃ overnight with the primary antibodies against ASC (CST, 67,824, 1:1000), pro-caspase-1 (Abcam, ab32499, 1:10,000), caspase-1 (Santa, sc-56036, 1:500), IL-1β (CST, 12,703, 1:1000), IL-18 (CST, 54,943, 1:1000), and GAPDH (Abcam, ab181602, 1:10,000). The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated antibodies at 23 °C for 1 h and then developed using ECL-Plus reagent (Thermo Fisher Scientific). We observed the membranes using gel imaging system and analyzed the findings.

Kidney tissue or cultured cell lysates were prepared with ice-cold lysis buffer (CST # 9806) containing protease inhibitor cocktail (cOmplete Mini, Roche #11836153001) and phosphatase inhibitor (PhosSTOP, Roche #4906837001), resolved on 8–12% gradient gels, transferred on to polyvinylidene difluoride membranes, and probed with the following antibodies: DPEP1 (Proteintech #12222-1-AP 1:500), CHMP1A (Proteintech #15761-1-AP 1:500), RIPK3 (Sigma #PRS2283 1:1000), Cleaved Caspase 1 (Santa cruz #sc-56036 1:500), Collagen III (Abcam #ab7778 1:1000), Fibronectin (Abcam #ab2413 1:1000), aSMA (Sigma #A5228 1:1000), ACSL4 (Abcam #ab155282 1:1000), CD63 (Abcam #ab193349 1:1000), GPX4 (Abcam #ab125066 1:1000), Actin (Sigma #A3854 1:20000), GAPDH (Proteintech #60004-1-Ig 1:1000), and Tubulin (BioLegend #801202 1:1000). Anti-rabbit IgG (H + L) (DyLight™ 800 4X PEG Conjugate) (CST #5151 1:10000) and Anti-mouse IgG (H + L) (DyLight™ 680 Conjugate 1:10000) (CST #5470) was used as a secondary antibody.