Caspase 1

Cells were washed using PBS and lysed using the protein lysis buffer (M-per) to obtain protein lysates. Equal amounts (30 μg) of proteins were loaded and separated using SDS-PAGE electrophoresis. The following primary antibodies were used: NLRP3 (Cell Signaling Technology, Danvers, MA, USA, 15101S, 1:1000), caspase-1 (Millipore, Burlington, MA, USA, AB1871, 1:1000), IL-1β (Cell Signaling Technology, 52718, 1:1000), β-Actin (Cell Signaling Technology, 4970S, 1:5000). β-actin was used as a loading control, and the density of bands was analyzed using the ImageJ software (NIH, Bethesda, MD, USA).
The IPs were performed as previously described [53 (link)]. Briefly, the lysates were preincubated with 1 μg rabbit anti-NLRP3 antibody (Cell Signaling Technology, D4D8T) overnight at 4 °C. Subsequently, 20 μL of protein G Dynabeads (Invitrogen, Carlsbad, CA, USA, 10003D) were added, followed by incubation at 4 °C for 4 h and centrifugation for 5 min at 3000× g. The pellet obtained was washed using RIPA buffer and resuspended in 2× loading buffer. Samples were boiled to elute the protein complexes from the beads. NLRP3 antibody (Cell Signaling Technology, 15101S) was used for normalizing the loading amount. IgG was used as the negative control.

Total protein was extracted from all animals using ice-cold RIPA buffer (Thermo Scientific, Rochester NY) containing a Halt^® protease inhibitor cocktail (Thermo Scientific, Rochester NY) and quantified using the Bio-Rad Protein Assay Reagent. Protein lysates (30 μg) were separated by SDS-PAGE gel and transferred onto nitrocellulose membranes, which were blocked with 5% skim milk at room temperature for 2 hours, and incubated overnight at 4 °C with the following antibodies: GAPDH (Santa Cruz, sc-25778), NLRP3 (R&D, 7578), ASC (Santa Cruz, sc-22514), Caspase-1 (Millipore, AB1871), and phospho-Smad2 (Cell Signaling, 3108). Following the overnight incubation, anti-rabbit IgG (Cell Signaling, 7074P2) and anti-mouse IgG (Cell Signaling, 7076P2) conjugated to horseradish peroxidase were used as secondary antibodies and signal was visualized with an enhanced chemiluminescence detection system (Bio-Rad, Rochester NY) and quantified by densitometry.

These studies were carried out according to a previously published procedure from our lab [39 (link)]. Astrocytic cell lysates were prepared using modified RIPA buffer and normalized for equal amounts of protein loading using a Bradford protein assay. Equal amounts of protein (30 µg) were loaded for each sample and separated using a 12% SDS-PAGE gel. Upon completion of the separation, the proteins were transferred to a nitrocellulose membrane and the nonspecific-binding sites were blocked using a blocking buffer. Membranes were then probed overnight at 4 °C with the respective primary antibodies pPKCδ, PKCδ, STAT3, pSTAT3, NLRC4 (1:1000, Rabbit monoclonal), GFAP (1:1000, mouse monoclonal) and ASC (1:1000, Rabbit polyclonal; EMD Millipore), Caspase-1 (1:1000, mouse polyclonal). Following incubation, the membranes were washed 3 times with PBS, and then the membranes were visualized using IRDye-tagged secondary antibodies on the Odyssey infrared imaging system (LI-COR); β-actin (1:10000, mouse monoclonal) was used as a loading control.

To assess the protein expression levels of TRPV4, NLRP3, Caspase-1, GSDMD, GSDMD-N, GSDMD-C, dynamin-related protein 1 (DRP1), mitochondrial fission factor (MFF), OPA1 mitochondrial dynamin like GTPase (OPA1), and mitofusin 2 (MFN2), lung tissue or 16HBE cells were lysed and equal amount of protein was subjected to 8 or 10% SDS-PAGE, subsequently, protein was transferred to 0.22 μm PVDF membranes (Merck-Millipore, Carrigtwohill, Ireland). The membranes were blocked with 5% non-fat milk powder and then incubated with antibodies overnight at 4°C: GAPDH, β-actin, DRP1, MFF, OPA1, MFN2 (1:1,000, Cell Signaling Technology, Danvers, MA, United States), Caspase-1, NLRP3 (1:1,000, RD system, Minneapolis, MN, United States), GSDMD, GSDMD-N, and GSDMD-C (1:1,000, Abcam, Cambridge, United Kingdom).
Membranes were then washed and incubated with HRP-conjugated donkey anti-mouse IgG or antirabbit IgG antibody for 1 h at RT. Finally, membranes were visualized with enhanced chemiluminescence (MerckMillipore, Carrigtwohill, Ireland). Quantitative images were analyzed using Image J software.

At the indicated time point, hMDMs in 12-well plates where washed with PBS and immediately lysed by adding 300 µl boiling Laemmli sample buffer and additionally boiled for 10 min. Samples were treated with benzonase (Merck) for 30 min at 37°C and equal amounts of lysates were separated by 8–16% SDS polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto PVDF membranes. The membranes were blocked with 5% milk powder in PBS Tween-20 (0.075%) and incubated with a primary antibody recognizing the p45 proenzyme (∼45 kDa) and the p20 subunit (∼20 kDa) of caspase-1 (1∶1000; Merck), followed by stripping and re-probing with β-actin (1∶10 000) as a loading control. The specific proteins were detected with a commercial enhanced luminol-based chemiluminescent (ECL) kit (Amersham). Densitometric analysis of bands was performed using NIH/ImageJ, and a caspase-1 p20/p45 ratio (cleaved active caspase-1/unprocessed inactive caspase-1) was constructed to evaluate caspase-1 activation in a way that was unbiased of difference in total protein across samples.

The chondrocytes and knee articular cartilage tissue were flushed three times with PBS and lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. The samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim-milk powder for 30 min at room temperature and then incubated with the corresponding primary antibodies of NLRP3, ASC, caspase-1, IL-6, IL-18, TNF-α, MMP13, ADAMTS4/5, IGF-1, aggrecan and Col2a1 (1:1000, Sigma-Aldrich, St. Louis, MO) at 4 °C overnight. After washing with PBS, the membranes were incubated with horseradish peroxidase-labelled secondary antibody (1:2000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature. Ultimately, the intensity of protein expression was measured by an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Carlsbad, CA). GAPDH served as control.