Standard polyacrylamide bis tris gel

Samples were heated to 75°C for 10 min then loaded into a standard polyacrylamide Bis-Tris gel (Invitrogen). SDS-PAGE was performed in MOPS running buffer (Invitrogen) at 175 V for 75 min. Protein was transferred onto a nitrocellulose membrane using the iBlot dry transfer system (Invitrogen). The membrane was blocked with Odyssey blocking buffer (LI-COR) for 1 h and incubated with a primary antibody in blocking buffer overnight at 4°C. The following day, the membrane was washed in 1× PBS containing Tween 20 (0.1%) and then incubated in blocking buffer containing either goat anti-rabbit or goat anti-mouse (LI-COR) IRDye 800CW secondary antibody at a concentration of 1:10,000 for 1 h at room temperature. Primary antibodies included the following: mouse anti-rat high-mobility group box 1 (HMGB1) monoclonal antibody (1:4000; Abcam); rabbit anti-rat nod-like receptor protein 3 (NLRP3) monoclonal antibody (1:1000; Millipore); and mouse anti-rat β-actin (1:200,000; Sigma-Aldrich). Protein expression was quantified using an Odyssey Infrared Imager (LI-COR) and normalized to the housekeeping protein value for that sample, and data are presented as the percentage of the within-blot regular diet control samples.