Anti sirt3

For the detection of antigens, the following primary antibodies and dilutions were used: anti-SIRT1 (1:100; cat. no. 13161-1-AP; Proteintech, Rosemont, IL, USA), anti-SIRT3 (1:200; cat. no. orb247889; Biorbyt, Cambridge, United Kingdom), anti-Na, K-ATPase 1 alpha1 (1:200; cat. no. NB300-146; Novus, Centennial, CO, USA), and anti-Connexin26 (1:200; cat. no. 710500; Life Technologies, Carlsbad, CA, USA). Fluorophore-conjugated secondary antibodies were used at a dilution of 1:500. Primary antibody labeling was performed at 4°C overnight after blocking in 10% goat or donkey serum for 10 min in PBS. Secondary antibody labeling was performed at 25°C for 1 h. Hoechst 33258 dye (Molecular Probes, Eugene, OR, USA) was used for 10 min to perform nuclear staining. The samples were examined under the BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

The total proteins were extracted from cell samples using RIPA lysate buffer containing PMSF and phosphatase inhibitor. The Pierce BCA protein assay kit (Thermo Fisher Scientific, Germany) was used to quantify proteins. Equal amounts of proteins were separated by SDS-PAGE, using 7% polyacrylamide gels and then transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 1 × TBS buffer containing 5% BSA and 0.05% Tween 20 for 3 h, incubated with primary antibodies at appropriate dilutions for 2 h and subsequently incubated with peroxidase-conjugated goat-anti-rabbit IgG (1:5000 dilutions) for 1 h. Immunoreactive proteins were visualized by using DAB staining (Beyotime, China). Primary antibodies used in this study included anti-mTOR (7C10), anti-p-mTOR (ser2448) (Cell Signaling Technology, Danvers, MA), anti-SIRT3, anti-GLS1, anti-GDH, anti-IDH2, anti-PGC-1α, anti-α-KGDH (Proteintech, USA), and anti-β-actin (Abcam, USA).