Neuronal extraction buffer

Neuronal extraction buffer (87792, Thermo Fisher Scientific) mixed with 1X protease inhibitor cocktail and 0.1 mM PMSF was used to solubilize cultured cortical cells and brain tissues. For protein quantification, BCA assay kit (BioVision), BioTek Synergy plate reader and Gen5 software (BioTek Instruments, Winooski, VT) were used. Immunoblotting was conducted using a 12 % SDS gradient polyacrylamide gel followed by transfer on PVDF membrane. The primary antibodies used in this paper are as follows: ATPase5β (1:1000, Sigma-Aldrich HPA001520), β-actin (1:5000, BioRad HCA147P), α-tubulin (1:2000, Sigma-Aldrich T6074), Sirt3 (1:1000, Cell Signaling Technology 5490S), and anti-acetyl lysine (1:1000, Cell Signaling Technology 9441S).

Cultured cortical cells and brain tissues were solubilized in the neuronal extraction buffer (87792; Thermo Fisher Scientific) supplemented with 1X protease inhibitor cocktail and 0.1 mM PMSF. Protein concentrations were quantified using a BCA assay kit (BioVision) and BioTek Synergy plate reader and Gen5 software (BioTek Instruments). Immunoblotting was conducted using 4–10% SDS gradient polyacrylamide gel followed by transfer on PVDF membrane. The following primary antibodies were used: Sirt3 (1:1,000, 5490S; Cell Signaling Technology, rabbit, reactivity for human, mouse, and rat), anti-acetyl-lysine (1:1,000, 9441S; Cell Signaling Technology rabbit, all species expected), ATPase5β (1:1,000, HPA001520; Sigma-Aldrich, rabbit, reactivity for rat, mouse, and humans), β-actin (1:5,000; HCA147P; Bio-Rad, Human Combinatorial Antibody Library/HuCAL, reactivity for rat, mouse, and humans), α-tubulin (1:2,000, T6074; Sigma-Aldrich, mouse, reactivity for mouse, chicken, Chlamydomonas, African green monkey, human, rat, bovine, sea urchin, and kangaroo rat). Blots were developed by chemiluminescence using ECL (34579; Thermo Fisher Scientific) and images were acquired on ChemiDoc XRS⁺ molecular imager (Bio-Rad). Band intensity was quantified using ImageJ.