Ab22684

The treated cells were lysed in RIPA buffer containing 1 mM DTT, 11 μg/ml DNase I, and protease inhibitor cocktail (Roche) and incubated on ice for 30 min. Sixty micrograms of protein sample were separated by electrophoresis on a denatured 10% SDS-PAGE gel and blotted onto a PVDF membrane (Millipore). The primary antibodies (Anti-Caspase-1, 1:1,000, ab138483, Abcam; Anti-cleavage Caspase-1, 1:1,000, ab207802, Abcam; Anti-Caspase-11, 1:1,000, ab22684, Abcam Anti-cleavage Caspase-11, 1:1,000, ab180673, Abcam) were incubated overnight at 4°C. Rabbit monoclonal to GAPDH (EPR16891, Abcam) was used as a loading control. Afterward, the membranes were rinsed and subsequently incubated with appropriate secondary antibodies. An ECL kit (Beyotime Biotechnology, Shanghai, China) was used to detect the bands of Western blots.

Proteins were drawn from the tissues and cells and quantified by using a BCA protein kit (Thermo Scientific). Proteins (50 μg) were loaded on SDS-PAGE gels per lane and transferred to PVDF membranes after electrophoresis. Then blocking membranes with 5% BSA at 2 h room temperature and incubating at 4°C overnight with the primary antibodies: GAPDH (Abcam, ab8245, 1:5,000), GSDMD (Santa Cruz Biotechnology, sc-393581, 1:1,000), caspase-11 (Abcam, ab22684, 1:1,000), caspase-1 (Abcam, ab138483, 1:1,000), caspase1 (p10), and caspase-1 (p20) (AdipoGen, AG-20B-0044, AG-20B-0042, 1:1,000). Immunoreactivity was detected by incubating with secondary antibodies (Abcam ab205718, ab97023, 1: 20,000).

Cells or heart tissues were lysed in ice-cold RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitors (Roche) for 30 min. Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with primary antibodies toward caspase-1 (Abcam, Cambridge, UK, ab138483), cleaved caspase-1 (Abcam, ab207802), caspase-11 (Abcam,ab22684), cleaved caspase-11 (Abcam, ab180673), USP46 (Abcam, ab88795), Tubulin (Cell Signaling Technology, Danvers, MA, USA, D65A4), GAPDH (Abcam, ab8245) and β-actin (Cell Signaling Technology, 8H10D10), respectively. After washing with PBS-Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and protein bands were visualized using Pierce^® ECL Western blotting substrate (Pierce, Rockford, IL, USA). Densitometric analysis was performed using Image J.