Anti il 1β

Total cell lysates were prepared using Laemmli buffer containing 5% β-mercaptoethanol. Protein concentrations were determined using Nanodrop ND1000 (Thermo Scientific). Proteins (20–50 μg) were boiled, separated on SDS–PAGE, and transferred onto PVDF membranes (Bio-Rad Laboratories). Membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST, Sigma-Aldrich) for 1 h at room temperature, and incubated overnight at 4 °C with the following primary antibodies diluted 1:1,000 in 5% BSA/PBST; anti-NLRP3 (#AG-20B-0014-C100, Adipogen), anti-IL-1β (#2022), anti-total Syk (#2712), anti-caspase-1 (#4199), anti-caspase-5 (#4429), anti-caspase-4 (#4450) (all from Cell Signaling) and anti-GAPDH (#MAB374, Millipore). For phospho-Syk detection, membranes were blocked and incubated with the primary antibody (#2710S, Cell Signaling) in 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST). Densitometry analysis was performed using ImageJ software and the data were normalized against GAPDH. Images have been cropped for presentation. Full-size blot images shown in Figs 1, 2, 3, 4, 5, 6, 7 and Supplementary Figs 1, 5, 8 and 9 are included in Supplementary Fig. 11.

Brain tissues and cultured cells were harvested and then lysed with RIPA buffer (Solarbio, China) containing cOmplete™ protease inhibitor cocktail (Roche). Proteins were electrophoretically separated on 10% SDS-PAGE gels and then transferred onto PVDF membranes (Millipore, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-PGC-1α (1:1000, Thermo Fisher), anti-LC3 (1:1000, Cell Signaling Technology), anti-ERRα (1:1000, Cell Signaling Technology), anti-ULK1 (1:1000, Cell Signaling Technology), anti-sequestosome1 (SQSTM1) (1:1000, Cell Signaling Technology), anti-TOMM20 (1:1000, Cell Signaling Technology), anti-COX IV (1:1000, Abcam), anti-IMMT (1:1000, Abcam), anti-NLRP3 (1:1000, AdipoGen), anti-ASC (1:200, Santa Cruz), and anti-IL-1β (1:800, AdipoGen). After washing, the specific blots were incubated with the species-appropriate secondary antibodies for 1 h at room temperature. Finally, the protein bands were viewed with a Gel Doc 2000 imaging system (Bio-Rad, USA) and then analyzed with the ImageJ software. In the quantitative analysis of Western blots, all the bands detected were within the linear range of detection.