Rabbit anti pdk1 antibody

An aliquot of the left ventricle was homogenised in ice‐cold buffer containing 50 mm Tris‐HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1% IGEPAL, 0.1% β‐mercaptoethanol and 10 μL mL^buffer−1 protease inhibitor cocktail. Tissue lysate was centrifuged at 13,000 g for 10 min at 4°C and the supernatant stored at −80°C. Homogenate protein content was determined using a bicinchoninic acid assay (Pierce, UK). Protein samples were run on a 12% Bis‐Tris acrylamide gel for ∼2 h at constant 100 V and transferred to a polyvinylidenedifluoride (PVDF) membrane for 2 h at constant 250 mA in a cooled transfer tank. The membrane was blocked for 1 h and incubated overnight at 4°C with rabbit anti‐PDK1 antibody (Cell Signaling, Leiden, the Netherlands), rabbit anti‐PDK2 antibody (Abgent, San Diego, CA, USA), rabbit anti‐PDK4 (Abgent, San Diego, CA, USA) and rabbit anti‐actin antibody (Sigma‐Aldrich, Gillingham, Dorset, UK). Membranes were then washed and incubated with goat anti‐rabbit HRP‐conjugated secondary antibody (R&D Systems, Abingdon, Oxon, UK). Washed membranes were incubated with enhanced chemiluminesence detection solution (Amersham, Bucks, UK) and exposed to X‐ray film (Kodak, Watford, UK).