A13687

Cells were lysed using RIPA buffer (Tris 20 mM, NaCl 150 mM, KCl 20 mM, MgCl₂ 1.5 mM, glycerol 10%, Triton X-100 1%, pH 7.5), and the protein concentration was measured by the BCA Protein Assay Kit (C503021; Sangon Biotech, Shanghai, China). Equal amounts of proteins were separated by 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene fluoride membranes, and the target proteins were finally detected using standard Western blotting protocols and visualized using the Super Signal West Pico Plus Luminol/Enhancer Solution (UC279012; Thermo, Waltham, USA). β-Actin was used as the loading control. The primary antibodies used were listed as follows unless otherwise specified: anti-β-actin antibody (AC038; ABclonal, Wuhan, China), anti-DLD antibody (A5220; ABclonal), anti-PDHA1 antibody (A13687; ABclonal), anti-DLAT antibody (A8814; ABclonal). The secondary antibodies used were as follows unless otherwise specified: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) (AS014; ABclonal).

Proteins were electrophoresed with 4%–20% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C in primary antibody diluent. Then, the membrane was incubated with secondary antibody for 1 h at room temperature. All bands were measured and analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA). The primary antibody was anti-PDHA1 (1 µg/ml, A13687, ABclonal, CHN). The secondary antibodies such as horseradish peroxidase (HRP)-conjugated anti-rabbit (A6154) and anti-mouse (A4416) antibodies were from Sigma-Aldrich.