Tissue or pellets were resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer (150 mM NaCl, 50 mM tris-HCl, 6 mM sodium deoxycholate, 1% NP-40, 1 mM EDTA, 1 mM NaF) supplemented with 1× protease inhibitor (Calbiochem) and 1× phosphatase inhibitor cocktails 2 and 3 (Millipore Sigma). Homogenates were allowed to equilibrate for 1 hour at 4°C, followed by centrifugation (13,000 rpm for 20 min) at 4°C. Supernatant was then collected followed by a protein assay. For immunoprecipitation of GRK2, GRK2 antibody conjugated to agarose was added to 1 mg of total protein and rotated for 1.5 hours at 4°C. Beads were washed three times with RIPA and solubilized in sample buffer. For immunoblotting, samples were prepared in sample buffer and (30 μg) loaded on a 4 to 20% gel and transferred on nitrocellulose membranes. The membrane was blocked in LI-COR block buffer for 1 hour at room temperature, followed by exposure to primary antibody at 4°C. Primary antibodies used were as follows: GRK2 rabbit (Santa Cruz Biotechnology, sc-562), GRK2 mouse (Millipore), rabbit phospho-670 GRK2 (Invitrogen), mouse VDAC (NeuroMab), GLUT1 rabbit (Cell Signaling Technology, 12939), GLUT4 mouse (Cell Signaling Technology, 2213), Gβγ rabbit (Santa Cruz Biotechnology, sc-261), HIF-1α (Cayman, 10006421), and β-actin mouse (Santa Cruz Biotechnology, 47778). Membranes were washed three times with PBS containing 0.1% Tween-20 and exposed to fluorescent conjugated secondary antibodies: rabbit 680 (Life Technologies, A21109), mouse 680 (Thermo Fisher Scientific, A21058), rabbit 800 (Cell Signaling Technology, 5151S), and/or mouse 800 (Cell Signaling Technology, 5257S). Imaging and quantification were done using LI-COR Odyssey software.

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