Anti mfn2 antibody

White adipose tissue samples were fixed overnight in 4% paraformaldehyde, after which they were sliced into 5-μm-thick sections. Cells grown on coverslips were fixed in 4% paraformaldehyde at room temperature and permeabilized with 0.1% Triton X-100 (Sigma, United States). For histologic examination, tissue slices were stained with hematoxylin. For immunofluorescence and immunohistochemistry, tissue slices or cells were incubated with blocking buffer (1% bovine serum albumin) for 1 h, and then with anti-UCP1 (Proteintech, China), anti-COX IV antibody (Proteintech, China), or anti-MFN2 antibody (Cell Signaling Technology, United States) at 4°C overnight. After that, the tissue slices or cells were incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibody (for immunofluorescence) or horseradish peroxidase–conjugated secondary antibody (for immunohistochemistry) at room temperature for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Zhongshan Jinqiao, ZLI-9557). Immunofluorescence images were captured with an AV300-ASW confocal microscope (Olympus America Inc., Center Valley, PA, United States).

Cortical neurons infected with scramble or Mu1 shRNA were treated with 10 uM MG132 (474790; Calbiochem) for 4 h. Cells were then lysed in RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) supplemented with protease inhibitor. The lysate was then incubated with control rabbit IgG (2729S; Cell Signaling) or anti-Mfn2 antibody (9842; Cell Signaling) at 4 °C overnight, and then incubated with protein A-Sepharose (17-0780-01; GE-Healthcare). The recovered immunocomplex was extensively washed with lysis buffer four times and eluted with reducing LDS sample buffer. Samples were resolved with 4–12% NuPAGE followed by immunoblotting with mouse anti-Ub (sc-8017; Santa Cruz), and rabbit anti-Mfn2 (11925; Cell Signaling).