Protein samples (30 µg/well) were loaded onto 10% SDS-PAGE to semi-quantify the changes in the regulatory enzymes and kinases of the cardiac metabolism as described previously [
37,
38]. The membrane was then incubated with different primary antibodies (1:1000), except for molecular weight > 100 kDa where (1:500) was used, and correspondent secondary antibodies (1:5000), based on the protein of interest. The following antibodies were used: with the following primary antibodies: mTOR (Cell Signaling, catalog 2983), phospho-mTOR (Cell Signaling, catalog 2983S), P70S6K (Cell Signaling, catalog 9205), phospho-P70S6K (Cell Signaling, catalog 9205S), SERCA2A (Cell Signaling, catalog 4388S), phospho-IRS-1 (Milipore, catalog 09-433), IRS-1 (Cell Signaling, catalog 2382S), phospho-Akt (Cell Signaling, catalog 9271S), Akt (Cell Signaling, catalog 9272S), phospho-GSK-3β (Cell Signaling, catalog 9322S), GSK-3β (Cell Signaling, catalog 9321S), phospho-AS160 (Cell Signaling, catalog 4288S), AS160 (Cell Signaling, catalog 2447S), GLUT4 (Cell Signaling, catalog 2213S), phospho-PDH (Milipore, catalog ABS204), PDH (Cell Signaling, catalog 3205S), BCATm (Cell Signaling, Catalog 9432S), KLF15 (Santa Cruz, catalog sc-271675), phospho-p38 (Cell Signaling, catalog 9211S), p38 (Cell Signaling, catalog 9212S), phospho-TAK1 (Cell Signaling, catalog 4508S), TAK1 (Cell Signaling, catalog 4505S), phospho-AMPK (Cell Signaling, catalog 2535S), AMPK (Cell Signaling, catalog 2603S), phospho-ACC (Milipore, catalog 07-303), ACC (Cell Signaling, catalog 3662S), MCD (Abcam, catalog ab95945), acetyl lysin (Cell Signaling, catalog 9441S), β-HAD (Abcam, catalog ab37673), LCAD (Abcam, catalog ab129711), BDH1 (Abcam, catalog ab68321), SCOT (Abcam, catalog ab70413). Membranes were then incubated with the appropriate secondary antibodies (goat anti-rabbit, catalog 7074P2; goat anti-mouse, catalog 31430; goat anti-chicken, catalog A16054) for 1–2 h. The protein bands were visualised using the Amersham enhanced chemiluminescence (Cell Signalling Technologies, Danvers, Massachusetts, USA). Densitometry was conducted in a blind fashion using ImageJ program (1.48v, National Institutes of Health USA) and all protein bands intensity were normalised to α-tubulin which served as an internal control (loading control).