Anti fabp4

Western blot analysis was performed as described previously^{37 (link)}. Briefly, samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes and incubated overnight at 4 °C with rabbit polyclonal anti-OB (1:1000; Millipore), anti-PPARγ (1:1000; Proteintech), anti-FABP4 (1:500; Proteintech) or mouse polyclonal anti-β-actin (1:10,000; Abcam) antibodies. Membranes were then incubated for 1 h at room temperature with fluorescence-labeled anti-mouse or anti-rabbit IgGs (1:5000) prior to visualization with an Odyssey Imager (LI-COR, Odyssey, NE, USA). Formalin-fixed adipose tissues were sectioned at 4 μm intervals, blocked with 1% BSA, and incubated overnight at 4 °C with anti-OB, anti-PPARγ and anti-PABP4 (1:1000 dilution for each antibody).

We lysed the cultured cells and homogenized tissues in RIPA buffer (Cat# P0045, Beyotime, China) containing a protease-inhibitor cocktail (Cat# 04693132001, Roche, Canada). After centrifugation at 14,000 rpm for 15 min at 4 °C, the protein concentrations were determined using a BCA protein assay kit (Cat# P0012, Beyotime, China). The cell and tissue lysates were then electrophoretically separated on 10% polyacrylamide gels and transferred onto PVDF membranes. The membranes were subsequently blocked with 5% nonfat dry milk dissolved in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature. After blocking, membranes were probed with the following diluted primary antibodies: anti-FASN (1:1000; Cell Signaling Technology, USA), anti-NOX4 (1:1000; Abcam, USA), anti-FABP4 (1:1000; Proteintech, China), anti-PPARγ (1:1000; Bioworld, USA), anti-β-actin (1:5000; Bioworld, USA) and anti-GAPDH (1:2000; Abcam, USA). After the membranes were incubated with the primary antibodies at 4 °C overnight, the membranes were incubated with goat anti-rabbit (1:1000, Beyotime, China) HRP-conjugated secondary antibodies and signals were detected using Image Lab software (Bio-Rad, USA).