Rabbit anti cleaved caspase 1

Proteins were loaded (50 μg), separated by 6, 8, and 15% SDS–PAGE and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The primary antibodies: rabbit anti-NLRC4 (1:800, Novus Biologicals, USA), mouse anti-pNLRC4 (1:400, ECM Biologicals, USA), mouse anti-RGS2 (1:500, Santa Cruz, USA), rabbit anti-Pro-caspase-1 (1:1000, Abconal, China), rabbit anti-Pro-IL-1β (1:1000, Abconal), rabbit anti-IL-18 (1:1000, Abconal, China), rabbit anti-cleaved caspase-1 (1:500, Cell Signaling, USA), rabbit anti-cleaved IL-1β (1:500, Affinity, USA), rabbit anti-cleaved IL-18 (1:200, R&D, USA), rabbit anti-TNF-α (1:500, Boster, China), rabbit anti-IL-6 (1:500, Boster, China), rabbit anti-LRRK2 (1:1000, Abcam, USA), rabbit anti-pLRRK2 s935 (1:500, Abcam, USA) and rabbit anti-β-actin (1:1000, Proteintech, USA). Secondary antibody (1:2000, Proteintech, USA)-linked horseradish peroxidase (HRP) was added to the bands for 2 h. ECL detection reagents (Thermo, USA) were used to visualize the bands. ImageJ software was used to determine the relative density of these proteins.

Cell lysates were obtained by lysing cells in 1% SDS lysis buffer containing 1% β-mercaptoethanol (Gibco). The lysates were immediately boiled for 10 min to minimize protein degradation. Supernatants were diluted in SDS buffer and boiled for 10 min prior to loading. Western blot analysis was performed using the following antibodies: rabbit anti-cleaved caspase-1 (89332S; Cell Signaling Technology), rabbit anti-gasdermin D (39754S; Cell Signaling Technology), rabbit anti-mouse cleaved caspase-8 (8592P; Cell Signaling Technology), mouse anti-RipK1 (610459; BD Biosciences), rabbit anti-RipK3 (2283; ProSci Inc), rabbit anti-phospho RipK3 (91702S; Cell Signaling Technology), rabbit anti-phospho MLKL (37333S; Cell Signaling Technology), rabbit anti-caspase-1 p10 (SC-514; Santa Cruz Biotechnology), mouse anti-caspase-1 (sc-56036; Santa Cruz Biotechnology), mouse anti-β-actin (SC-81178; Santa Cruz Biotechnology), goat anti-mouse IgG HRP (172-1011; Bio-Rad Laboratories Inc), and goat anti-rabbit IgG HRP (A6154; MilliporeSigma). The blots were subsequently detected by chemiluminescence with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific Inc) on a ChemiDoc MP imager (Bio-Rad Laboratories Inc).

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).