Western Blot Analysis of Inflammatory Markers

Western blot analysis of cell and tissue lysates was performed as previously described [13 (link)]. Briefly, membranes were blocked with 5% nonfat dry milk in TBST for 1 h and incubated overnight at 4 °C with primary Abs against SOD2 (Cell Signaling, Danvers, MA), NLRP3, ASC, pro-caspase-1, cleaved caspase-1, ACTB and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary Ab (Cell Signaling), and protein bands were detected by chemiluminescence using Immobilon Forte Western HRP substrate (Millipore WBLUF0500). Densitometry was performed using ImageJ software (NIH, Bethesda, MD). All experiments were performed in triplicate.