P2x7r

In this experiment, proteins were isolated from chondrocytes. In short, cells were firstly rinsed in PBS, then placed in RIPA buffer for lysis, and centrifuged for collection of protein. Prior to SDS/PAGE, quantification of protein was performed using the BCA Protein Assay Kit (Thermo Fisher Scientific). Subsequently, a mixture of proteins was isolated via SDS/PAGE, and protein bands were transferred on a polyvinylidene fluoride membrane. Primary antibody of P2X7R (1 : 1000; Abcam), which was diluted in accordance with the experimental requirements, was added on the membrane for incubation at 4 °C for 24 h. Afterwards, the membrane was EDTA incubated with secondary antibody for 2 h. Finally, with an ECL kit, the bands on the membrane were visualized. In this procedure, protein expression of β‐actin (1 : 1000; Abcam) was used as the internal control.

For immunoblot analyses, modified RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) was used to extract total protein from frozen lung tissue [24 (link)]. Extraction proteins from tissues and cells were measured using the BCA protein assay reagent kit (Pierce). An equal amount of total protein (80 μg of protein/lane) was resolved on a 5–12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% nonfat dry milk in PBST (containing 0.05% Tween 20). Incubation with the antibodies was performed using the following dilutions: 1:750 for P2X₇R (Abcam, USA) and 1:1000 for caspase-1, procaspase-1, caspase-1, IL-1β (all of them from Cell Signaling Technology, USA) and total NLRP3 (Biosource, Belgium). Primary antibodies were detected with horseradish peroxidase-conjugated antibodies, 1:5000 for anti-mouse (ZSJQ-BIO, Beijing, China) and 1:5000 for anti-rabbit (ZSJQ-BIO, Beijing, China), at room temperature for 1.5 h. Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualised using a FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA). Blot bands were qualified using NIH ImageJ software.