Total protein isolation and Western blotting were performed as described previously [ref]. The blots were incubated with antibody an anti-CISD1 (1:2000) (Protein Tech, 16006-1-AP), an anti-β-actin (1:5000) (Sigma-Aldrich, St Louis, MO, A5441), an anti-phospho-IkBα (1:1000) (Santa Cruz Biotechnology, sc-8404), an anti-IkBα (1:1000) (Santa Cruz Biotechnology, sc-847) and an anti-COX2 (1:1000) (Cayman chemical, 160106), an anti-iNOS (1:1000) (Santa Cruz Biotechnology, sc-650) and an anti-HO-1 (1:3000) (Enzo Life Sciences, ADI-SPA-896), an anti-SOD2 (1:3000) (Santa Cruz Biotechnology, sc-30080), an anti-SOD1 (1:1000) (Santa Cruz Biotechnology, sc-11407) and anti-NLRP3/NALP3 (1:1000) (Adipogen, AG-20B-0014-C100), an-anti-P2X7R (1:3000) (Alomone Labs, APR-004) in TBST overnight at room temperature. The blots were incubated with an anti-secondary antibody (1:5000) in TBST, immunoblots were detected by SuperSignal® West Pico Chemiluminescent Substrate (Pierce) and visualized after exposure to X-ray film.
Mouse IL-1β (R&D systems, Minneapolis, MN, USA, DY401), IL-6 (R&D systems, Minneapolis, MN, USA, DY406), and TNFα (R&D systems, Minneapolis, MN, USA, DY410) were measured from cell culture supernatant of BMDMs and RAW264.7 cells using enzyme-linked immunosorbent assay (ELISA).
Mouse IL-1β (R&D systems, Minneapolis, MN, USA, DY401), IL-6 (R&D systems, Minneapolis, MN, USA, DY406), and TNFα (R&D systems, Minneapolis, MN, USA, DY410) were measured from cell culture supernatant of BMDMs and RAW264.7 cells using enzyme-linked immunosorbent assay (ELISA).