Abs204

Western blotting experiments were carried out as previously described^{48 (link)}. Anti-HSP90 (610419; BD Biosciences), anti-MPC1 (ab74871; Abcam), anti-phospho-PDHE1α (Ser-293) (ABS204; Millipore) and anti-PDHE1α (#3205; Cell Signaling Technology) antibodies were used at a dilution of 1/1000 (or 1/250 for the anti-MPC1 antibody) in 5% bovine serum albumin (BSA). Anti-MCT1 and anti-MCT4 antibodies (custom-made, ThermoFisher Scientific) were used at 1/1000 in 5% skimmed milk. Uncropped versions of Western blots are presented in Supplementary Fig. 7.

Heart tissue samples were homogenized with a Bioprep-24 (Hangzhou Allsheng Instruments Co., Ltd.) in T-PER buffer (#78510, Thermo Fisher Scientific, Waltham, MA, USA), protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), and phosphatase inhibitor cocktail (Roche Diagnostics). To collect supernatant, tissue lysates were centrifuged at 10,000 rpm for 10 min at 4 °C. Subsequently, 5 μg of protein were loaded in each lane of a 10% Bis–Tris gel (Thermo Fisher Scientific) and separated using SDS-PAGE and electroblotted to nitrocellulose membranes using an iBlot2 Dry Blotting System (Thermo Fisher Scientific). Membranes were blocked with Setsuyakukun supporter (DRC, Tama, Tokyo, Japan) for 1 h at room temperature, washed, and incubated for 2 h at room temperature in Kiwami Setsuyakukun buffer with anti-phospho-PDH antibodies (ABS204 and ABS194, Millipore), anti-total-PDH antibodies (ab110334, Abcam, Cambridge, United Kingdom), anti-PDK4 antibodies (ab89295, Abcam), or anti-GAPDH antibodies (AM3400, Thermo Fisher Scientific). Target proteins were detected with appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and a chemiluminescence kit (#170-5060, Bio-Rad Laboratories). Signals were detected with a ChemiDoc Touch (Bio-Rad, Hercules, CA, USA), and quantitative analysis was performed using Image Lab (Bio-Rad).

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).