Quantitative Protein Analysis by Western Blotting

Gel electrophoresis and western blotting were performed using the Xcell Surelock system (Invitrogen) using precast gradient gels (4–20%) as described [68] (link). The following antibodies were used: Anti-PDH-E1α (pSer293), Merck Millipore 1:10,000; and Anti-PDH-E1α, Abcam, 1:10,000. For visualisation of Western blots, HRP-based secondary antibodies were used followed by chemiluminescent detection on Kodak X-Omat film. Western blots were analysed by digitally scanning the blots, followed by densitometric analysis (ImageJ). For figure preparation of example western blots, linear adjustment of brightness/contrast was applied (Photoshop) equally across the image.