Sirt2

Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (Thermo). Nuclear and cytoplasmic components were isolated using NE-PER nuclear and cytoplasmic extraction kit (Thermo). Homogenates (20 μg of total protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes.
Antibodies were used against the following proteins: Sirt6, Sirt1, Ac-H3K9, FoxO1, p-FoxO1 (Cell Signaling, Beverly, MA, USA), Ac-lysine, Sirt2, Sirt3 (Abcam, Cambridge, UK), ubiquitin (Santa Cruz Biochemicals, Dallas, TX, USA), Sirt4, lamin B, GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7, Ac-FoxO1 (LifeSpan Biosciences, Seattle, WA, USA), and HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA).

Whole cell lysates were collected using RIPA buffer with added protease inhibitor cocktail (Roche). Insoluble fractions were collected using Urea-containing buffer. Protein was quantified using Bradford assay reagent (Biorad) and bovine serum albumin standards (Pierce). Equal amounts of protein were electrophoresed in SDS-PAGE gels and transferred to PVDF membrane Immobilon-FL (Millipore). Blots were developed using ECL-plus (GE) for 5 minutes, chemiluminescence visualized on a Licor Odyssey FL imager and quantitation done using Licor Image Studio software. All quantitated protein signals were normalized to GAPDH signal from the same gel. Antibodies used were as follows: SIRT1 (Cell Signaling), SIRT2 (Abcam), SIRT3 (Sigma), Involucrin (Santa Cruz), Cytokeratin 10(Santa Cruz), GAPDH (Santa Cruz), p-NBS1 Ser 343 (Cell Signaling), NBS1 (Santa Cruz), ac-p53 (Cell Signaling), p53 (Calbiochem), pATM Ser 1981 (Cell Signaling), ATM(Cell Signaling), pCHK2 Ser 19 (Cell Signaling), CHK2(Cell Signaling).

GP17 (molecular weight = 947.154; purity > 98%) was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). The positive control drug NBP was obtained from CSPC NBP Pharmaceutical Co., Ltd. Triphenyltetrazolium chloride (TTC) was purchased from Sigma–Aldrich (MO, United States). Primary antibodies against p62/SQSTM1, LC3-B, BNIP3, NAMPT, SIRT1, SIRT2, MnSOD, PGC-1α, FOXO3 and p-FOXO3 were obtained from Abcam (Cambridge, UK). Primary antibodies against Hif1α and Beclin1 were obtained from Proteintech (Wuhan, China). A primary antibody against SIRT3 was obtained from Cell Signaling Technology (MA, USA). The inducer RAP and the inhibitors 3-MA and 2-ME were obtained from MedChemExpress (New Jersey, USA). The inhibitor AGK-7 was obtained from Abcam (Cambridge, UK). ELISA kits for IL-6, TNF–α, MCP-1, T-AOC and 4-HNE were acquired from HaiTai TongDa Sci Tech, Ltd. (Beijing, China).

Antibodies for cleaved-caspase 3 (Asp175), caspase 3, phospho-AMPK (Thr172), AMPK, cyclin D1, acetylated-lysine, phospho-Akt (Ser473), phospho-GSK-3β (Ser9), phospho-cdc2 (Tyr15), cdc2, c-myc, phospho-Erk1/2 (Thr202/Tyr204) were purchased from Cell Signaling. β-catenin, Sirt-1, Sirt-2 were purchased from Abcam. PGC1α was purchased from Millipore. β-actin was purchased from Sungene. Whole cell lysate was prepared using RIPA lysis buffer in the presence of protease inhibitors. Protein concentrations were determined using the bicinchoninic acid (BCA) method (Biomed, Beijing, China). Total cell lysates were separated using 8%-12% SDS-PAGE, transferred onto PVDF membranes, and then detected using various primary antibodies. The antibody-antigen complexes were detected using the Chemiluminescent HRP Substrate (Millipore, MA, USA).
For immunoprecipitations, cells were lysed in lysis buffer (50 mM Tris-HCl at pH 7.4, 0.2 mM EDTA, 150 mM NaCl, 3% NP-40, 1 mM phenyl methylsulfonyl fluoride (PMSF) and, Protease inhibitor cocktail). Equal amounts of cell extracts were incubated with anti-PGC1α antibodies. Subsequent immunoblots were performed as described above.

Protein expression was examined by Western blot analysis. A total of 50 μg protein was separated by SDS-PAGE (12% polyacrylamide gel) and transferred to a PVDF membrane. The blots were blocked for 3 hours at room temperature with Tris buffer solution (TBS) containing 5% nonfat dry milk and 0.05% Tween 20. The membranes were incubated overnight at 4°C with rabbit primary polyclonal antibodies (SIRT 1, Santa Cruz Biotechnology, Santa Cruz, CA; SIRT 2, SIRT 3, and PPAR-γ, Abcam) at a final dilution of 1 : 1000. Then, the membranes were incubated for 2 h at room temperature with a secondary antibody (goat anti-rabbit horseradish peroxidase conjugated, dilution 1 : 10,000, Santa Cruz Biotechnology, Santa Cruz, CA). After incubation, the blots were visualized using a chemiluminescence kit (Immobilon Western, Millipore, MA, USA). Blots were stripped and reincubated with monoclonal α-actin antibody as control. Images from films were digitally acquired by GS-800 densitometer with the Quantity One software (Bio-Rad). The values of each band density are expressed as arbitrary units (AU).

Antibodies used included: SIRT2 (Abcam, Cat. No. ab211033), CTGF (Abcam, Cat. No. ab6992), FN1 (ABclonal, Wuhan, China; Cat. No. A12977), COL1A1 (Santa Cruz, Cat. No. sc-59772), TGF-β1 (Abcam, Cat. No. ab92486), β-actin (bioss, Beijing, China; Cat. No. bs-0061R), COL3A1 (Santa Cruz, Cat. No. sc-514601), E-cadherin (BD Biosciences, Cat. No. 610182), α-SMA (Santa Cruz, Cat. No. sc-84326), p-SMAD2 (Cell Signaling Technology, Cat. No. 18338), SMAD2 (Abcam, Cat. No. ab40855), acetyl Lysine (Abcam, Cat. No. ab190479), Ubiquitin (Novus, Cat. No. NB300-129), SMURF2 (ABclonal, Cat. No. A2278), CREBBP (ABclonal, Cat. No. A14237), P/CAF (ABclonal, Cat. No. A0066), Goat Anti-Rabbit IgG H&L (HRP) (Abcam, Cat. No. ab6721), and goat anti-mouse IgG H&L (HRP) (Abcam, Cat. No. ab205719).