Anti sirt2

Cells were lysed on ice for 30 min, using cold RIPA buffer supplemented with protease inhibitor tablets (Pierce). After centrifuged at 10,000 × g for 30 min at 4 °C, cleared lysates were stored at −80 °C until used for immunoblotting. Samples were run on a 4–12% SDS-PAGE gel prior to transfer to nitrocellulose membranes. Anti-V5 (Invitrogen, Cat# R960-25), anti-Flag (Sigma, Cat#: SAB4200071), anti-acetyl-Lys (Cell Signaling Technology, Cat#: 9441), anti-myc (Covance, Cat# 904401), anti-HA or anti-HA-HRP (Roche, cat#: 11867423001, 12013819001), anti-SIRT1 (Santa Cruz Biotechnology, Cat#: sc-74504), and anti-SIRT2 (Cell Signaling Technology, Cat#: 12650) were used to detect the proteins of interest while anti-actin (Sigma, Cat#: SAB4301137), anti-GAPDH (Millipore, Cat#: AB2302), and anti-tubulin (Abcam, Cat#: AB14128) were used as loading controls. The blots were probed with the indicated primary antibodies followed either by Alexa Fluor-conjugated secondary antibodies for near-infrared fluorescence detection or by HRP-conjugated secondary antibodies for chemiluminescence detection. Unsaturated signals were collected using Hyperfilm ECL (GE healthcare), Odyssey Fc (Li-Cor) or iBright FL1500 (ThermoFisher) imaging system. Full scan of cropped immunoblots were provided in Supplementary Fig. 1.

Total protein was isolated from cells using cold radio immunoprecipitation assay buffer containing a protease inhibitor cocktail (Roche Applied Science, Penzberg, Bavaria, Germany). A total of 50 μg of protein was subjected to SDS-PAGE, and the blots were transferred onto a polyvinylidene difluoride membrane. After blocking, the membranes were incubated with anti-SIRT1 (Millipore, Temecula, CA, USA), anti-SIRT2 (Cell Signaling Technology, Danvers, MA, USA), anti-SIRT3 (Cell Signaling Technology), anti-SIRT5 (Millipore), anti-SIRT6 (Cell Signaling Technology), anti-eNOS (Cell Signaling Technology), anti-KLF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FOXM1 (Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology), and anti-FLAG (Sigma-Aldrich) antibodies. Antibodies against cell cycle regulators and checkpoint molecules, including cyclin D1, cyclin D3, p18 INK4C, p21 Waf1/Cip1, p27 Kip1, CDK2, CDK6, phospho-RB, and phospho-p53 (Ser15), were purchased from Cell Signaling Technology. Immune-reactive protein bands were visualized by chemiluminescence using ECL reagents (GE Healthcare, Fairfield, CT, USA). Protein expression was imaged in a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).

Unless otherwise noted, all chemical reagents were purchased from Sigma–Aldrich (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM) was purchased from Life Technologies. Ethylene diamine tetraacetic acid (EDTA) free protease inhibitor was purchased from Roche Applied Science (Germany). Pre-stained protein ladder was purchased from Bio-Rad (Hercules, CA). Pre-cast polyacrylamide gels (4–12% NuPAGE Bis-Tris gels) were purchased from Life Technologies. Mass spectrometry grade trypsin was purchased from Promega (Madison, WI). High capacity streptavidin beads were purchased from ThermoFisher Scientific (Waltham, MA). Antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) (anti-Sirt1, anti-Sirt2, and anti-γ-actin antibodies), Cell Signaling Technology (Danvers, MA) (anti-Sirt3, anti-HSP60, anti-fibrillarin, and anti-histone H3 antibodies), Abcam (United Kingdom) (anti-H3K4Ac and anti-H3K4Me3 antibodies), or PTM BioLabs (Chicago, IL) (anti-H3K4Cr, anti-H3K27Cr, and pan anti-crotonyllysine antibodies). Anti-Sirt3 N-term antibody was a generous gift from Dr Danny Reinberg (New York University, New York, United States).

Total cellular proteins were extracted from CD4+T cells and spleen mononuclear cells using a protein extraction kit (Keygen Biotech, Nanjing, China). Proteins were separated by 10% SDS/PAGE and transferred to PVDF membranes (Merck Millipore, USA). Membranes were washed three times with TBST, then blocked in TBST containing 5% BSA for 2 h at room temperature. Membranes were then incubated overnight at 4°C with anti-Sirt2, anti-HIF1a, anti-mTOR, anti-LDHA, anti-Glut1, anti-HK2, anti-PKM2 antibody (1:1000 dilution; Cell Signaling Technology) and anti-GAPDH(1:1000; Santa Cruz). Membranes were washed in TBST three times, and then incubated with goat anti-Rabbit IgG secondary antibody (1:5000 dilution; Santa Cruz) solution for 2 h at room temperature. Finally, protein bands were visualized using a chemiluminescence western blot detection system (Alpha Innotech; MicroChemi 4.2).

Whole cell protein extracts were prepared according to Laemmli (Laemmli 1970 (link)). The primary antibodies used were the following: anti-ATM (1:500), anti-phospho-ATM Ser1981 (1:500), and anti-GAPDH (1:50,000) (Millipore, Warsaw, Poland); anti-p53 (1:500), anti-p21 (1:500), and anti-p16 (1:250) (Santa Cruz Biotechnology, Santa Cruz, USA); anti-phospho-p53 Ser15 (1:250) (Becton Dickinson, Warsaw, Poland); anti-HO-1 (1:1000) (Enzo Life Sciences, Warsaw, Poland); SIRT1 (1:250), phospho-SIRT1 Ser47 (1:250), anti-SIRT2 (1:1000), anti-SIRT3 (1:500), anti-SIRT5 (1:500), anti-SIRT6 (1:1000), anti-SIRT7 (1:250), anti-phospho-p38 Thr180/Tyr182 (1:500), and anti-p38 (1:500) (Cell Signaling Technology, Poznań, Poland); and anti-AGTR1 (1:500) and anti-actin (1:50,000) (Sigma-Aldrich, Poznan, Poland). The respective proteins were detected after incubation with one of the horseradish peroxidase-conjugated secondary antibodies (1:2000) (Dako, Gdynia, Poland), using an ECL system (Thermo Scientific, Warsaw, Poland), according to the manufacturer’s instructions.