Rabbit anti acc

Isolated cardiomyocytes were lysed in a lysis buffer containing: 50 mM β-glycerol phosphate, 2 mM EGTA, 1 mM DTT, 10 mM NaF, 1 mM sodium orthovanadate, 20 mM HEPES (pH 7.4), 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail tablet. Protein levels of AMPK-α, p-AMPK, ACC, p-ACC, and p-cTnI were examined by standard western immunoblotting (Cui et al., 2013 (link); Tong et al., 2013 (link)). Membranes were probed with anti-rabbit AMPK-α (1:1000, Cell Signaling), anti-rabbit phosphor-AMPK (Thr¹⁷², 1:1000, Cell Signaling), anti-rabbit ACC (1:1000, Cell Signaling), and anti-rabbit phosphor-ACC (Ser⁷⁹, 1:1000, Cell Signaling), followed by incubation with horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody (Cell Signaling Technology Inc, Beverly, MA). For the detection of cTnI Ser¹⁴⁹ phosphorylation, a polyclonal antibody was generated against the phosphopeptide LRRVRIS(phos)ADAMMQA and purified with affinity cross-absorption with the nonphosphorylated peptide (Sancho Solis et al., 2011 (link)). Blue X-ray film (Phenix, Candler, NC) was used for photon detection and image development. Films were scanned with the Bio-Rad GS-700 scanner in the core facility of the School of Medicine and Biomedical Sciences and the relative density of the bands on the film was determined by Image J software.