Pro caspase 1

Proteins were extracted from liver tissue or cells with ice‐cold lysis buffer (50 mm Tris, 150 mm NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1% Triton X‐100). Proteins (20 μg/sample) were subjected to 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride nitrocellulose membranes (Bio‐Rad, Hercules, CA, USA). Monoclonal antirabbit NLRP3, cleaved caspase‐1, procaspase‐1, ASC, IL‐1β, pro‐IL‐1β, phospho‐AMPK, AMPK, phospho‐mTOR, mTOR, p62, LC3B, Bcl‐2, Bcl‐xL and β‐actin antibodies (Cell Signaling Technology) were used.

Total and cytoplasmic protein was isolated from retina samples. Proteins were run on 10 or 12 % polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were blocked with 5 % BSA at RT for 60–90 min and incubated overnight at 4 °C with antigen-specific primary antibodies. Blots were then incubated with species-specific HRP-conjugated secondary antibodies for 60 min at RT. Proteins were visualized by incubation with a chemiluminescence substrate kit (ECL Plus; Perkin Elmer Inc., Covina, CA, USA). The expression of target proteins was quantified by Quantity One software (The Discovery Series) after normalizing to β-actin or GAPDH.
The primary antibodies and dilutions were used as follows: NLRP3 (1:500; NBP1-77080, Novus, Littleton, CO, USA, 100 kD), ASC (1:500; 04–147, Millipore, Temecula, CA, USA, 22 kD), phosphor-NF-κB p65 (1:1000; #3033, Cell Signaling Technology, Beverly, MA, USA, 65 kD), cleaved caspase-8 (1:500; #8592, Cell Signaling Technology, Beverly, MA, USA, 18 kD), caspase-1 (1:200; AB1871, Chemicon, International, Inc., USA, pro-caspase-1 45 kD, cleaved-caspase-1 20 kD), IL-1β (1:500; #8689, Cell Signaling Technology, Beverly, MA, USA, pro-IL-1β 31 kD, IL-1β 17 kD), β-actin (1:1000; MAB1445, MultiSciences Biotech, Hangzhou, China, 45 kD), and GAPDH (#2118, Cell Signaling, Boston, MA, USA, 36 kD).

Snap-frozen whole lung samples were homogenized in phosphate buffer (150 mM NaCl, 50 mM Tris–HCl, 1% Triton X-100, pH 7.4, 1 mM EDTA, 0.1% SDS, and 1% sodium deoxycholate) with protease inhibitors and centrifuged at 12,000 rpm at 4 °C for 10 min to obtain cytosolic protein fractions. Equal amounts of protein were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were incubated at 4 °C overnight with the following primary antibodies: GPX4 (1:200, sc-58607), FTH1, NOX4, TLR4, apoptosis-associated speck-like protein containing a caspase (ASC), pro-caspase1, caspase1, and NLRP3 (1:1000; all Cell Signaling Technology, USA). All antibodies were incubated overnight at 4 °C in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST). The membranes were washed and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; ZSGB-Bio, Beijing, China) for 1 h at room temperature. The membranes were developed with enhanced chemiluminescence reagents, protein bands were visualized by exposure to X-ray film using a gel imaging system (UVP, Upland, CA), and the protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Band intensity was normalized to GAPDH levels.

Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing proteinase and phosphatase inhibitors. A BCA assay kit (Beyotime) was used to measure the concentration of protein. The supernatant proteins were precipitated. The protein of lytic samples was transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, China) after separating by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime). Membranes were incubated with antibodies against gasdermin D (GSDMD, Abcam, Cambridge, UK), IL-1β (Abcam), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc-1, Abcam), TRAP (Abcam), pro caspase-1 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-1 (Cell Signaling Technology), ASC (Cell Signaling Technology), TNF receptor-associated factor (TRAF) 2 (Proteintech, Rosemont, IL, USA), TRAF6 (Proteintech), c-Fos (Proteintech), cathepsin K (CTSK, Proteintech), matrix metalloprotein 9 (MMP9, Proteintech), and anti-actin (Beyotime) overnight at 4 °C after blocking with quick block buffer (Beyotime) for 30 min. Then, membranes were washed three times with TBS-Tween and incubated with the appropriate secondary antibody for 1 h at room temperature. The results were visualised via chemiluminescent peroxidase substrate (Proteintech).

Total samples lysates containing approximately equal amounts of proteins were solubilized in sodium dodecyl sulfate (SDS)‐sample buffer by heating at 95°C for 10 minutes, separated by 12% SDS‐polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes and incubated with primary antibodies against AIM2, ASC (Santa Cruz Biotechnology), pro‐Caspase‐1, Caspase‐1, Pro‐IL‐1β, IL‐1β, GAPDH (Cell Signaling Technology) overnight at 4°C. After washing with Tris‐buffered saline containing 0.1% tween‐20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)‐linked secondary antibody (Southern Biotech, Birmingham, AL, USA) for 2 hours at room temperature and rinsed with TBST. Then, the immunoreactive bands were detected by chemiluminescence using the ECL (Thermo Fisher Scientific).