Anti immt

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.

Specific antibodies were purchased from the following commercial sources for the indicated experiments: anti-CS (ab129095, 1:2,000 for immunoblotting (IB)), anti-IMMT (ab137057, 1:1,000 or IB), anti-ANP (ab191398, 1:1,000 for IB, 1:50 for immunofluorescence), anti-BNP (ab92500, 1:10,00 for IB), anti-NFATC4 (ab62613, 1:1,000 for IB, 1:50 for IF), anti-MLC2v (ab92721, 1:50 for IF), goat anti-rabbit IgG H&L (Alexa Fluor 594) (150080, 1:250 for IF), goat anti-mouse IgG H&L (Alexa Fluor 488) (150113, 1:250 for IF) from Abcam. Additionally, anti-Tom20 (42406, 1:2,000 for IB, 1:50 for IF), anti-Vinculin (13901, 1:2,000 for IB), anti-OPA1 (67589S, 1:1,000 for IB, 1:50 for IP) from Cell Signaling Technology; anti-α-actinin (A7811, 1:200 for IF) were purchased from Sigma. From Abclonal, we purchased anti-ATP5B (A5769, 1:1,000 for IB), and anti-MLC2a (17283-1-AP, 1:50 for IF) from Proteintech. Anti-DYKDDDDK-tag (M20008, 1:10,000 for IB), anti-GAPDH (M20050, 1:5,000 for IB) were purchased from Abmart, and HRP goat anti-mouse IgG (H+L) (BK-R050, 1:5,000 for IB) and HRP goat anti-rabbit IgG (H+L) (BK-M050, 1:5,000 for IB) were purchased from Bioker.