Sirt1 antibody

Sodium hydrosulfide (NaHS, a donor of H₂S) was purchased from Sigma (Sigma, St. Louis, MO, USA). Pelltobarbitalum Natricum was obtained from Germany and the Sirt1-antibody was purchase from American Abcam. The primary antibodies of CPR78, CHOP, cleaved caspase-12, Bax and Bcl2 were bought from cell signaling technolgy. The malondialdehyde (MDA) assay kit was bought from Uscn Life Science Inc. (Wuhan, Hubei, China). The glutathione (GSH) enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bio-Swamp Life Science. Total SOD assay kit and Bicinchoninic Acid (BCA) Protein Assay Kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Specific monoclonal antibody for detecting Sirtinol was purchased from Santa. Bicinchoninic Acid (BCA) Protein Assay Kit was obtained from Beyotime Institute of Biotechnology (Shanghai, China).

Co-immunoprecipitation assay was conducted using an immunoprecipitation kit (Abcam) as per the manufacturer's recommendations. In brief, BV-2 cells were transfected for 24 h with sh-USP22 or pcDNA3.1-KLF3-AS1. Cell lysate was prepared using RIPA buffer (Thermo). Each sample was incubated with Sirt1 antibody (Abcam) or USP22 antibody (Abcam) at 4 °C overnight. Afterwards, protein A/G Sepharose beads were added before incubated for 4 h at 4 °C. Protein-bead complex was eluted with NuPAGE LDS Sample Buffer (Life Technologies). The immunoprecipitated fractions were detected using western blot with anti-Sirt1 (Abcam), anti-ubiquitin (Abcam), anti-USP22 (Abcam) or anti-Tubulin (Abcam).

Cells were grown to confluence in 6 cm petri dishes, differentiated for 9 days (peak adipogenesis in the second AIM induction) and 21 days (endpoint of adipogenic induction) using the adipogenic protocol with and without NAM treatment and harvested in CellLytic lysis buffer (Sigma-Aldrich) with 0.01% protease inhibitor (Sigma-Aldrich) added. Cell lysate was sonicated and centrifuged; the supernatant was flash frozen and stored at -80°C until assay.
SIRT1 protein was extracted from cell supernatant using immunoprecipitation. Briefly, a 1:50 dilution of SIRT1 antibody (Abcam) was added to each sample of cell supernatant and incubated overnight at 4°C with agitation. The antibody-supernatant mixture was added to a 50% slurry of protein A agrose bead (Cell Signaling) and incubated for 3 hours at 4°C with agitation. SIRT1 protein activity was then measured by fluorometric assay at 350 nm (Abcam) according to the manufacturer’s protocol for quantification of SIRT1 activity.
Total SIRT1 protein was measured simultaneously by Simple Western size-based protein assay (WES, ProteinSimple, Santa Clara, CA) following manufacturer’s protocol. Results from WES were analyzed using ProteinSimple Compass software. SIRT1 activity in each cell set was normalized to the cell set’s total SIRT1 protein as measured by WES.

Etoposide (cat# E1383), Nicotinamide (cat# 479865-U) and Sirtinol (cat# s7942) were from Sigma-Aldrich Company Ltd (Dorset, UK). Cisplatin (cat# 440-040) and metothrexate (cat# 440-045) were from Enzo Life Sciences UK Ltd (Exeter, UK). EX527 (cat# 2780) was from Tocris bioscience (R&D systems, UK). Tissue cell culture media were supplied by Gibco Life Technologies and foetal calf serum by Harlan Seralab (UK). Cyclophilin A (cat# Ab3563), actin (cat# Ab8226), SIRT1 antibody (cat# Ab13749), and HRP-anti-mouse (cat# Ab6808) antibodies were from Abcam (UK). p53 BC-12 antibody (cat# SC126) was from Santa-Cruz Biotechnology (Texas, USA). c-Myc antibody (cat# 9402), Bcl-2 (cat# 2870) and HRP-anti-rabbit (cat# 7074) antibody were from Cell Signalling Technology (MA, USA). miR-34a mimic (cat# C-300551-07), control (cat# CP001000-02-05) were purchased from Dharmacon (now GE Healthcare, UK). siRNAs targeted for p53 (cat# 1299001) and scrambled siRNA were from Invitrogen.

Immunofluorescence was performed to determine the gene expressions, as described in our previous studies.¹⁷ The hippocampus was fixed with 4% paraformaldehyde for 24 h, cryoprotected with 30% sucrose for 48 h, and sectioned using a cryostat (Cryotome E, Thermo Fisher, Waltham, MA, USA). Coronal sections (10 μm thickness) were incubated with ARF6 antibody (1:100 dilution; Abcam, Cambridge, UK), SIRT1 antibody (1:50 dilution; Abcam, Cambridge, UK), or MMP2 antibody (1:200 dilution; Abcam, Cambridge, UK) overnight at 4°C, followed by incubation with a goat anti‐rabbit conjugated CY3 antibody (1:300 dilution; Servicebio, Wuhan, China) for 50 min at room temperature. Nuclei were subsequently counterstained with DAPI (Servicebio, Wuhan, China) for 10 min at room temperature. Images were captured using a Nikon Eclipse Ti confocal microscope. Hippocampal subregions CA1 and DG were analyzed for ARF6, SIRT1, and MMP2 expressions.

MPS was purchased from Pfizer and dissolved in sterile normal saline. VO-OHpic was purchased from selleck (USA, S8174) and dissolved in DMSO. And the final concentration of DMSO is the same (0.1%) in each group including the control. CD31 antibody (UK, ab182981), CD34 antibody (UK, ab81289), VEGF Receptor 2 (VEGFR2) antibody (UK, ab11939), VEGF Receptor 1 (VEGFR1) antibody (UK, ab32152), CD133 antibody (UK, ab19898), SIRT-1 antibody (UK, ab189494), NQO-1 antibody (UK, ab80588), and Trx antibody (UK, ab133524) were purchased from Abcam. FITC-conjugated mouse anti-rabbit IgG antibody was purchased from Boster (China, BM2012). Bad antibody (USA, #9268, for IP), Phospho-Bad antibody (USA, #5284), Bcl-xL antibody (USA, #2764), AKT antibody (USA, #4691), p-AKT antibody (USA, #4060), cleaved caspase-3 (Asp175) antibody (USA, #9661), cleaved caspase-9 antibody (USA, #9508), MFF antibody (USA, #84580), and normal rabbit IgG (USA, #2729) were purchased from Cell Signaling Technology. Bax antibody (USA, 50599-2-Ig, for western blot), Cytochrome C antibody (USA, 66264-1-Ig), Nrf2 antibody (USA, 16396-1-AP), Bcl-xL antibody (USA, 26967-1-AP), Bad antibody (USA, 10435-1-AP), FIS1 antibody (USA, 10956-1-AP), VEGF antibody (USA, 19003-1-AP), HO-1 antibody (USA, 10701-1-AP), and GAPDH antibody (USA, 60004-1-Ig) were purchased from Proteintech.