Western Blot Analysis of PDH Signaling

Western blot analyses were performed as previously described [99 (link)]. Briefly, cells were lysed in 1% triton-X buffer supplemented with a phosphatase inhibitor (P5726), protease inhibitor (P8340) and leupeptin trifluoroacetate (L2023). Lysates were centrifuged, and protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad 500-0116). Equivalent amounts of protein were loaded on a 12% resolving acrylamide gel. Protein transfer was conducted overnight at 4 °C using a nitrocellulose membrane (0.45 µM, Thermo 88018, Thermo Fisher Scientific). The membranes were blocked with 5% BSA in TBS and washed (TBST). Blocked membranes were labeled with primary antibody overnight at 4 °C. Primary antibodies were labeled with near-infrared secondary antibodies (IRDyes 680 RD or 800 CW, LI-COR Biosciences, Lincoln, NE, USA), detected and quantified using Odyssey Fc Imaging System (LI-COR Biosciences). Primary antibodies were: phospho-PDH (ABS204, MERCK, Darmstadt, Germany), total-PDH (C54G1, cell signaling), antibeta ACTIN (A1978), and antialpha TUBULIN (T9026).