Cpt1a

Protein lysates were loaded on a polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane using the mini trans-blot transfer system (Bio-Rad). To detect specific antigens, blots were probed with primary antibodies CPT1A (Cell Signaling, 12252 Dilution 1:1000) GAPDH (Santa Cruz, sc-32233 Dilution 1:5000), and CPT1C (abcam ab123784 Dilution 1:500) on a shaker at 4 °C, overnight, followed by 1 h of room temperature incubation with HRP-conjugated secondary antibodies (Santa Cruz). Chemoluminescence was recorded using the automated Gel Doc XR system (Azure).

Primary antibodies including CPT1A (Cell signaling), PPAR-γ (Santa Cruz Biotechnology), β-catenin, β-actin (Millipore), p-AMPK(T172)/AMPK (Cell signaling), p-AKT(T308)/Akt (Cell Signaling) were used at 1:1000. Secondary antibodies including goat anti-rabbit IgG-HRP (Millipore) and goat anti-mouse IgG-HRP (Millipore) were used at 1:5000. Cells were lysed in Laemmli sample buffer after treatment and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis. For immunoprecipitation, cells were lysed in radio immunoprecipitation assay (RIPA) buffer [10 mM sodium phosphate (pH 8.0), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS in the presence of 1 mM DTT, 1 mM phenylmethylsulfonylfluoride, and a protease inhibitor cocktail (Sigma)], precleared with protein A beads, and then incubated with 1 μg of antibody against β-catenin or isotype control IgG with protein A-agarose beads on a rotator overnight at 4°C. After 3 washes with RIPA buffer, immunoprecipitated complexes were eluted in sample buffer by boiling and subjected to immunoblot analysis. Immunoblots were visualized by chemiluminescence substrate (ThermoFisher) and imaged by a ChemiDoc XRSplus system (BioRad).