Pierce enhanced chemiluminescence ecl system

After priming with gp120 for 48 h, microglial cells were treated with Meth for another 24 h. The cells were then lysed using RIPA buffer (Bio-Rad, Hercules, CA) for analyzing pro-IL-1β processing. 30 μg total proteins were separated by 4–20% gradient PAGE and transferred to nitrocellulose polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in tris-buffered saline (TBS) and incubated overnight at 4°C with either rabbit polyclonal antibody to IL-1β at a 1:500 dilution (Abcam, Boston, MA), mouse polyclonal antibody to NLRP3 at a 1:1000 dilution (Adipogen, San Diego, CA), rabbit Iba-1 antibody at 1:1000 dilution (Fujifilm, Richmond, VA) or anti-mouse β-actin monoclonal antibody (1:5000, Sigma-Aldrich). The washing buffer was TBS with 0.2% Tween (TBS-T). The secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibody (1:10000, Jackson Immuno Research Laboratories, PA). Labeled proteins were shown by the Pierce-enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA).