Ab200577

For immunofluorescence, deparaffinised sections were incubated in the antigen unmasking solution (10 mM tri-sodium citrate, 0.05% Tween-20) for 8 min at 75°C, and treated with a blocking solution (3% BSA in PBS) for 30 min. The primary antibody (anti-BCKDHA, diluted 1:50 in PBS, rabbit polyclonal ab90691, Abcam; Cambridge, United Kingdom; anti-BCKDHA (phospho S293), diluted 1:50 in PBS, rabbit polyclonal ab200577, Abcam; anti-ELOVL6, diluted 1:200 in PBS, rabbit polyclonal ab69857, Abcam) was applied and the sections were incubated in a humidified chamber overnight at 4°C. Then, the sections were incubated for 1 h at room temperature with a conjugated secondary antibody (anti-rabbit IgG-FITC antibody produced in goat, diluted 1:100 in PBS, F0382, Sigma-Aldrich, St. Louis, MO, United States). Nuclei were stained with Hoechst Stain Solution (diluted 1:1000 in PBS, Hoechst 33258, Sigma-Aldrich). The slides were treated with PermaFluor Mountant (Thermo Fisher Scientific, Inc., Waltham, MA, United States) and cover slipped. The images were captured using a Leica Confocal Microscope TCS SP8 (Leica Microsystems). The staining intensity was expressed as the mean value pixel intensity (PI) using the software Leica application suite advanced fluorescences software.

Protein from frozen mouse hearts were prepared by lysis in ice-cold RIPA buffer (ThermoFisher, cat.89900) containing protease and phosphatase inhibitor (ThermoFisher, cat. A32959). Tissue was homogenized using bullet blender bead lysis kit (Next Advance), and protein concentrations were determined with Pierce BCA Protein Assay Kit (Thermofisher, cat. 23225). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4–15% gradient gel (Bio-Rad, cat.4561086) and transferred to PVDF membrane using iBlot 2 transfer system (ThermoFisher). Protein expression was measured by chemiluminescence using ChemiDoc imaging system (Bio-Rad). Proteins were detected with the following primary antibodies: BCKDH (ThermoFisher, cat.PA5-97248), phospho-BCKDH (S293) (abcam, ab200577), PP2Cm (abcam, ab135286), 4-hydroxynonenal (abcam, ab46545) (14 (link), 56 (link), 57 (link)).