Atp5a

To detect acetylation of mitochondrial proteins, immunoprecipitation assay was performed using anti-Acetyl-Lysine (mAb mix) affinity beads. Cortical neurons were seeded at 20 × 10⁶ in a T75 flask and treated with control, SIRT2 WT or KO OL-CM for 24 hours on DIV6-7. Neurons were collected on DIV7-8, washed with PBS, and lysed by lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 5% glycerol) and protease inhibitor cocktail (Roche) on ice for 30 min. Samples were centrifuged at 14, 000 g at 4° C for 15 min. Cell lysate (1 mg) was incubated at 4° C for 3 hours with Anti-Acetyl-Lysine (mAb mix) affinity beads (Cytoskeleton, Inc) that were coated with a mixture of two anti-acetyl lysine antibodies (Clones 7B5A1 and 10C4B2.1), then spun down and washed three times by cold lysis buffer. 50 μL 2 × sample buffer with 20 mM DTT was added into each sample and boiled for 5 min prior to loading for immunoblots. Primary antibodies were used as follows: ANT1 (1:500, Sigma), ANT2 (1:500 Cell Signaling), ATP5A (1:1000, Santa Cruz), NDUFA5 (1:1000, Thermo), PHB2 (1:1000, Proteintech). VeriBlot for IP detection reagent (1:2000, Abcam) was used as a secondary antibody to avoid IgG signals in the IP samples.