Nbp1 49533

For detection of ChAT, GLUT-4 and cardiac troponin I (cTnI, a cardiac marker), seven micrometer thick sections were probed with anti-ChAT (Abcam, AB181023), anti-GLUT-4 (NovusBio, NBP1-49533) and biotin-conjugated anti-cTnI antibodies (NovusBio, NB110-2546B) in a sequential manner.
For microvascular analysis, seven micrometer thick sections were probed with biotin-conjugated Isolectin-B4 (Vector laboratories, B1205; 1:200) and anti-α-smooth muscle actin conjugated with Cy3™ (Sigma-Aldrich, C6198; 1:800) to detect endothelial cells and smooth muscle cells, respectively. The vascular density was expressed as the mean number of Isolectin⁺ cells (for capillaries) or αSMA⁺ and Isolectin⁺ cells (for arterioles) per mm² of cardiac tissue.
To determine the level of fibrosis, sections were stained with 0.1% Direct Red 80 (Sigma Aldrich, 3,665,548)/Picric acid solution (Sigma Aldrich, 197,378). The fibrotic area was normalized to total tissue area and expressed as fold change relative to the control group.

Cells lysed with RIPA buffer were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electrotransferred onto polyvinylidene fluoride membranes. After overnight incubation with primary antibody at 4 °C, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit (Sigma-Aldrich, St. Louis, MO, USA) secondary antibody for 1 h at room temperature, followed by addition of ECL prime (Vazyme, Nanjing, China) to detect bands using a Bio-Rad gel documentation system (Bio-Rad, CA, USA). Primary antibodies were detected against Nephrin (ab80298, Abcam), Podocin (P0372, Sigma), Synaptopodin (NBP2-39100, Novus), GLUT1 (ab115730, Abcam), GLUT4 (NBP1-49533, Novus), HK2 (NBP2-02272, Novus), F6PK (ab154804, Abcam), PKM1 (7067 S, CST), p-PKM2 (3827 S, CST), PKM2 (4053 S, CST), PKLR (ab137787, Abcam), LDHA (3558 S, CST), PGC-1α (ab54481, Abcam), TFAM (ab131607, Abcam), OPA-1 (ab42364, Abcam), DRP-1 (ab184247, abcam), VDAC (4866 s, cst), PCNA (ab18197, abcam), p-S6 (4858 s, cst), S6 (2217 s, cst), and Tubulin (T9026, Sigma) at a dilution of 1:1000. Quantification was performed by measurement of the intensity of the signals with the use of ImageJ (NIH, Bethesda, MD, USA).