Anti sirt2

For cell‐based experiments, cells were washed three times in PBS and resuspended with lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P‐40, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride (PMSF), and 25 mM NAM and trichostatin A (TSA) for 20 min and centrifuged for 20 min at 13,800 g; the insoluble fraction was discarded. The lysates were fractionated by SDS/PAGE and transferred to nitrocellulose filter (NC) membranes. The membranes were blocked for 1 h with Tris‐buffered saline with Tween 20 (TBST) containing 5% (mass/vol) nonfat dry milk. After incubation with primary antibodies (anti‐acetylated tubulin (K40) (Proteintech), anti‐α‐tubulin (Invitrogen), anti‐SIRT2(Abcam) or anti‐His (Abmart), each diluted 1:1000), the membranes were washed and incubated with HRP‐conjugated anti‐mouse (Cell Signaling Technology) or anti‐rabbit secondary antibodies (Cell Signaling Technology), followed by detection using ECL Western blotting substrate (Bio‐Rad).

H9C2 cells with different treatment were lysed, quantified and subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Cat # 1,620,177, Bio-Rad, Hercules, CA, USA) which were incubated with 5% nonfat milk in TBST and followed by incubation of primary antibodies at 4°C overnight. Then the PVDF membranes were washed with TBST and incubated with secondary antibodies. Protein expression was detected using BeyoECL Moon (Cat # P0018FS, Beyotime) under a Tanon 5200 machine (Shanghai, China). The detailed primary and secondary antibodies were listed as below: anti-cleaved caspase 3 (Cat # E83-77, Abcam); anti-caspase 3 (Cat # ab32351, Abcam); anti-Bax (Cat # WL01637, Wanleibio, Shenyang, China); anti-Bcl-2 (Cat # WL01556, Wanleibio); anti-β-actin (Cat # WL01372, Wanleibio); anti-Nrf2 (Cat # WL02135, Wanleibio); anti-Sirt2 (Cat # ab211033, Abcam); anti-HO-1 (Cat # EP1391Y, Abcam); anti-GST (Cat # ab138491, Abcam); anti-NQO1 (Cat # 11,451-1-AP, Proteintech, Wuhan, China); anti-FOXO3a (Cat # 66,428-1-Ig, Proteintech); anti-GCLM (Cat # 14,241-1-AP, Proteintech); anti-Keap1 (Cat # 10,503-2-AP, Proteintech). IgG(H + L)(HRP-labeled Goat Anti-Rabbit IgG(H + L)) and IgG(H + L)(HRP-labeled Goat Anti-Mouse IgG(H + L)) were purchased from Beyotime (Beijing, China). All protein expressions were normalized by β-actin expression.

Western blotting was performed according to standard western blotting procedures. The harvested SH-SY5Y Cells were lysed in NP-40 buffer containing protease inhibitor cocktail (Sigma, USA) and 1 mM phenylmethylsulfonyl fluoride (Sigma, USA). Lysates were centrifuged at 12,000 g for 15 minutes at 4°C. Supernatants were collected, and protein concentrations were determined by the BCA Protein Assay Kit (Thermo, USA). Proteins were then separated via 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: a-tubulin (Abcam; 1:300) and anti-SIRT2 (Abcam; 1:1000). The proteins were visualized with enhanced chemiluminescence reagents (Pierce, Shanghai) in the machine (Azure Biosystems, USA).