Rabbit anti caspase 1

The following antibodies were used: rabbit anti-TXNIP (Cell Signaling, #14715), rabbit anti- PERK (Cell Signaling, #5683), rabbit anti-IL-18 (Abcam, ab68435), rabbit anti-NLRP3 (Abcam, ab214185), rabbit anti-IL-1β (Santa Cruz, sc-7884), rabbit anti-caspase-1 (Cell Signaling, #2225), mouse anti-Gasdermin D (Abcam, ab57785), mouse anti-β-actin (Cell Signaling), rabbit anti-BiP (Abcam, ab21685), rabbit anti-IRE1a (Abcam, ab48187), rabbit anti-phospho-MLKL (Ser358) (Cell Signaling, D6H3V), rat anti-MLKL (Millipore, MABC604), mouse anti-ASC (B-3) (Santa Cruz, sc-514414), rabbit anti-cleaved gasdermin D (Asp275) (Cell Signaling, #36425), goat anti-rabbit or mouse HRP-conjugated IgG (Cell Signaling), Alexa Fluor 488 donkey anti-rabbit or mouse IgG (Molecular Probes, A-21206), and Alexa Fluor 594 donkey anti-mouse IgG (Molecular Probes, A-21203). The following reagents were used: STF 083030 (IRE1a inhibitor, Santa Cruz, CAS 307543-71-1); resveratrol (TXNIP inhibitor, Sigma, R5010-100MG); PERK inhibitor II (Sigma, 5046510001); NLRP3 inhibitor (Sigma, 5381200001).

About 50 mg of liver and kidney samples were homogenized and centrifuged (13,000 g, 10 min, 4°C). Supernatants were collected and the protein content was determined using a BCA protein assay following the manufacturer’s instructions. 50 µg of total proteins were loaded for immunoblot experiments. Proteins were separated by either 8% or 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane, which was then incubated with primary antibodies (dilution 1:1000). The antibodies used were: rabbit anti-Thr¹⁸⁰/anti-Tyr¹⁸² p38 (Cell Signaling #9211); rabbit anti-total p38 (Cell Signaling #9212); mouse anti-NRLP3 (Adipogen- AG-20B-0014-C100); rabbit anti-Caspase-1 (Cell Signaling #24232); rabbit anti-Tyr³⁹⁷ FAK (Cell Signaling #3283); rabbit anti-total FAK (Cell Signaling #3285); mouse anti-Tyr⁴⁰² PyK2 (Cell Signaling #3291); mouse anti-total PyK2 (Santa Cruz #sc-393181); rabbit anti-β-actin (Cell Signaling #4970). Blots were then incubated with a secondary antibody (Cell Signaling - anti-mouse #7076; anti-rabbit #7074) conjugated with HRP (dilution 1:10000) and developed using the ECL detection system. The immunoreactive bands were analyzed by the Bio-Rad Image Lab SoftwareTM 6.0.1 and results were normalized to sham.

Protein was extracted from cultured H9c2 cells and rat heart tissue. The protein concentration was determined by a detergent-compatible Bradford protein assay kit (Beyotime, Nantong, China). Protein samples were subjected to 8% and 12% SDS–PAGE and blotted to a nitrocellulose filter membrane. The blots were blocked with 5% BSA, followed by incubation with the indicated primary antibodies, including mouse anti-β-actin, rabbit anti-Smad2, rabbit anti-phospho Smad2, rabbit anti-Gasdermin D, rabbit anti-cleaved Gasdermin D, rabbit anti-Caspase 1, rabbit anti-cleaved Caspase 1 (1:1000, Cell Signalling Technology), rabbit anti-α-SMA, mouse anti-COL3A1 and mouse anti-FN1 (1:500, Santa), overnight at 4°C. The membranes were then washed three times with TBST and incubated with secondary antibodies, including HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (diluted 1:10,000, ICL Lab, USA), for 2 hours at room temperature. The membranes were washed three times with TBST, and ECL western blotting detection reagent (Millipore) was used to develop the immunoblots. Protein bands were visualized by a GE Amersham Imager 800 RGB (EG, USA).