Ab209845

Total protein was isolated from MPC5 cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and quantitated using a BCA protein quantification kit (Abcam, Cambridge, UK), and the proteins were separated via 10% SDS‒PAGE. Then, the proteins were transferred to PVDF membranes (Roche, Basel, Switzerland). After being sealed with 5% nonfat milk and washed three times with TBST, the membranes were incubated with primary antibodies against Bax (1:1,500, ab182733; Abcam, Cambridge, UK), Bcl-2 (1:2,000, ab182858; Abcam, Cambridge, UK), α-SMA (1:400, MA5-14084; Thermo Fisher Scientific, Waltham, MA, USA), podocin (1:800, PA5-79757; Thermo Fisher Scientific, Waltham, MA, USA), synaptopodin (1:1,000, ab259976; Abcam, Cambridge, UK), p-p65 (1:800, MA5-15160), NLRP3 (1:800, ab263899), cleaved caspase-1 (1:1,000, PA5-99390; Thermo Fisher Scientific, Waltham, MA, USA), GSDMD (1:900, ab209845; Abcam, Cambridge, UK), and β-actin (1:8,000, MA1-140; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. After being washed three times with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1:7,000) for 1 h, and then the immunoblots were visualized using an ECL kit (Roche, Basel, Switzerland) and quantitated using ImageJ software (Bethesda, Rockville, MD, USA).

For signaling blots, the supernatant was removed, and cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors plus 4× Laemmli sample buffer. Caspase and GSDMD cleavage were measured from the combined cell lysate and supernatants. Proteins were separated via SDS-PAGE with 8 to 12% polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore), and blocked with 5% nonfat dry milk. Primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), caspase-3/cleaved-caspase-3 (9662 and 9661, CST), caspase-7/cleaved-caspase-7 (9492 and 9491, CST), caspase-8/cleaved-caspase-8 (4927, CST and AG-20T-0138-C100, Adipogen), caspase-9 (9504, CST), GSDMD (ab209845, Abcam), p-RIPK3 (91702, CST), RIPK3 (2283, ProSci), p-MLKL/MLKL (37333 and 37705, CST), and β-actin (4970, CST) were incubated overnight at 4°C followed by appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) incubated for 1 h at room temperature (Jackson ImmunoResearch, West Grove, PA). Membranes were visualized using Luminata Forte Chemiluminescence substrate (WBLUF0500, Millipore) or SuperSignal West Femto substrate (34096, Thermo Fisher Scientific) on a Bio-Rad ChemiDoc.

Cells were lysed in RIPA lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, 2 mM ethylenediaminetetraacetic acid [EDTA], 200 nM aprotinin, 20 μM leupeptin, 50 mM phenanthroline, and 280 mM benzamidine-HCl) and supernatant fractions were collected. Protein concentrations were measured with bicinchoninic acid (Pierce, Rockford, IL, USA), and equal amounts of proteins (50 μg/lane) were separated by 13% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to the nitrocellulose membranes (GE Healthcare Life Science, Pittsburgh, PA, USA) incubated with a specific antibody. The anti-caspase-1 antibody (3019-100; BioVision, Milpitas, CA, USA, and AG-20B-0042; AdipoGen, San Diego, CA, USA) and anti-GSDMD antibody (ab209845; Abcam PLC) were used as primary antibodies. These antibodies were used at a dilution of 1:1,000. The anti−ACTB antibody (A5441) was acquired from Sigma−Aldrich (Merck KGaA) and used at a dilution of 1:5000. Secondary antibodies (anti−rabbit IgG [1:2000 diluted; sc−2004] and anti−mouse IgG [1:2000 diluted; sc−2005]) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Bands were detected using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Darmstadt, Germany).