Acat1

Following tissue homogenization and determination of protein concentration, equal amounts of proteins (12 μg/well) were loaded in each well of a 4–20% SDS PAGE gel and transferred onto a 0.2 μm pore size nitrocellulose membrane. Membranes were blocked in 5% blocking solution (Bio-Rad Laboratories, Inc. Hercules, CA) in 0.01% PBS-Tween and immunoblotted overnight (4°C on shaker) with different primary antibodies. Blots were washed with 0.01% PBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ThermoFisher, Waltham, MA) for 1 h at room temperature. After incubation, membranes were washed before visualization using enhanced chemiluminescence (SuperSignal West Femto, ThermoFisher Scientific, Waltham, MA). Primary antibodies used were all rabbit polyclonal: BDH1 (Proteintech, 1:1,000); ACAT1 (Proteintech, 1:1,000); GLUT1 (Proteintech, 1:1,000); GLUT3 (Abcam, 1:2,000); COX IV (Abcam, 1:1,000) and β-actin (Invitrogen, 1:5,000). Band intensities were quantified by densitometry analysis using ImageJ software; corrected for background intensities and normalized to levels of β-actin (soluble fraction) or cytochrome oxygenase IV (COX IV, for mitochondrial enzymes) used as loading controls.

The expression of proteins was quantified with a BCA Protein Assay Reagent Kit (Thermo Fisher, USA). The rabbit polyclonal secondary antibody was purchased from Cell Signaling Technology (Boston, USA). GAPDH, LOXL2, VEGFA, CPT1A, ACSL1, and ACAT1 antibodies were purchased from Proteintech Group, Inc. (Chicago, IL, USA). The band density was quantified using ImageJ software.