Hexokinase 2

Tissue homogenates/lysates were normalized (90 µg of total protein per lane) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (NuPAGE; 4–12% Bis Tris-gradient gels, Invitrogen, Carlsbad, CA), transferred to nitrocellulose membranes, and analyzed by immunoblotting [2 (link)] (four rats/group). A total of four gels (labeled Gel 1 –4) were used to resolve all of the samples collected. After transfer, membranes were cut into strips representing desired molecular weight ranges based on a protein standard loaded in the far left lane of each gel [denoted as molecular weight marker (MWM) in kDa], and probed for protein levels by Western blotting. Primary antibodies against cas-pase-1, GAPDH, aldolase-A, and hexokinase II (Santa Cruz Biotechnology, Dallas. TX) were used. Protein transfer was assessed by staining membranes with BLOT Fast Stain (G Biosciences, St. Louis, MO), and total protein levels determined by fluorescence (Odyssey, LI-COR, Lincoln, NE). Total signal from each lane was used to normalize all immunoblot data.

Whole hearts were homogenized as previously described [Xing et al., 2003 (link)]. Briefly, the frozen hearts were homogenized with a Polytron (Brinkman Instruments) in lysis buffer, lysates were centrifuged at 13,000 × g for 10 min, supernatants were collected, and the protein concentrations were determined by the Bradford assay. The lysates were separated by SDS-PAGE, immunoblotted with primary and secondary antibodies, and immunoblots were developed using ECL reagents (PerkinElmer, USA). The protein bands were scanned by ImageScanner (Amersham Biosciences) and quantitated by densitometry (Fluorchem, 2.0; Alpha Innotech, San Leandro, CA). Antibodies purchased from commercial sources included: p-ACC-Ser⁷⁹ and AMPK α2 from Upstate; p-AMPK-Thr¹⁷², p-Akt-Thr³⁰⁸, p-Akt-Ser⁴⁷³, Phospho (Ser/Thr) Akt substrate (PAS) from Cell Signaling Technology; AS160 from Millipore; Hexokinase II and anti-α-Tubulin from Santa Cruz Biotechnology; and GLUT1 and GLUT4 from Chemicon International. Anti-SNARK and phospho-SNARK-Thr²⁰⁸, and p-AS160-Ser⁷¹¹antibodies were generated by Cell Signaling Technology. Horseradish peroxidase-conjugated anti-rabbit and anti-goat antibodies (Amersham Biosciences) were used to bind and detect all primary antibodies.