Rabbit anti sirt1

Western blotting was performed as described in the previous studies [33 (link),34 (link),35 (link)] with minor modifications. Protein samples were separated via 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore) for 3 h at 300 mA. The membranes were blocked with 5% nonfat dry milk or BSA dissolved in Tris–HCl saline buffer containing 0.1% Tween-20 (TBS-T, PH 7.4). Subsequently, the blots were incubated overnight at 4°C with one of the following antibodies: rabbit anti-p-AMPK (1:500; cat. no. ab23875; Abcam, USA) and rabbit anti-sirt1 (1:500; cat. no. ab220807; Abcam, USA). Membranes were washed three times for 5 min each time in TBS-T. HRP-coupled goat antirabbit secondary antibodies (1:1,000; Boster, Wuhan, China) diluted in TBS-T were then applied for 1 h. Membranes were washed three times in TBS-T for 5 min each time at room temperature. Immunoreactive signals were then visualized with the enhanced chemiluminescence solution (Bio-Rad, USA). Signal intensities were quantified by densitometric analysis using ImageJ software (Dental Diagnosis Science, San Antonio, TX).

Western blot analyses were performed as previously described (Bates et al., 2010 (link); Li et al., 2011c (link)), using an actin band for transfection cell lysates, to check equal loading across all lanes (Pendyala et al., 2010 (link)). The antibodies used were mouse anti-Bcl2 (1:1000, 692, Abcam Inc., Cambridge, MA), rabbit anti-Psen1 (1:500, 71181;Abcam), rabbit anti-Onecut2 (1:500, 28466;Abcam), rabbit anti-SIRT1 (1:500, 110304;Abcam), and rabbit anti-β-actin (1:1000, 8226;Abcam). Goat anti-mouse (31403, Thermo Fischer Scientific, Barrington, IL) was used for Bcl2 (1:2000), and goat anti-rabbit (31460, Thermo Scientific) for β-actin, SIRT1, Psen1, and Onecut2 (1:1000) as secondary antibodies. Intensities of antibody reactive bands were detected by Enhanced Chemiluminescence (ECL), (Pierce Biotechnology, Rockford, IL), and quantified by densitometry using ImageJ software (Public domain, NIH, USA).

CEP samples were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin, and then were sectioned from the side of nucleus pulposus (NP) or annulus fibrosus (AF) to bone side via length cutting (4 μm). This technique was supported by the Tissue Embryology Department of Chongqing Medical University. The sections were deparaffinized with xylene and rehydrated using graded ethanol and distilled water. Next, the sections were treated with 3% H₂O₂ for 15 minutes at room temperature to eliminate endogenous peroxidasesactivity, incubated with trypsin for 30 minutes at 37 °C to retrieve the antigen, and blocked with normal goat serum for 15 minutes at room temperature. Then, the sections were incubated with rabbit anti-SIRT1 (Abcam, USA, 1:200), rabbit anti-p53 (Cell signal, USA, 1:200), rabbit anti-p21 (Cell signal, USA, 1:200) and rabbit anti-p16 (Ruiying Biological, China, 1:150) primary antibodies overnight at 4 °C. The sections were then incubated with the secondary antibody goat anti-rabbit IgG-HRP. The resulting sections were photographed under a microscope (Leica, Germany). Five fields which distributed in the whole section were counted (400X), and the average positive rate was counted. Three sections were assessed per patient, and three patients were assessed per group (DDD and LVF).

Oxymatrine (purity >98%) was purchased from Sigma-Aldrich. Hepatic TG and FFA assay kits were from Nanjing Jiancheng Bioengineering Institute. Sequencing-grade trypsin was from Promega. iTRAQ reagent-8 plex multiplex kit was obtained from Applied Biosystems. Triple TOF 5600 mass spectrometer and Eksigent nanoLC-1D plus liquid chromatography were from SCIEX. Rabbit anti-Plin2, rabbit anti-L-FABP, rabbit anti-FASN, rabbit anti-Sirt1 and mouse anti-SCD1 monoclonal antibodies were from Abcam. Rabbit anti-AMPKα (phospho-Thr172) and rabbit anti-AMPKα monoclonal antibodies were from Cell Signaling Technology.

Cells were fixed with InsideFix (Miltenyi, Bologna, Italy; Cat. No. 130-090-477) and incubated with the primary antibody rabbit anti-SIRT1 (1:80; Abcam, Cambridge, United Kingdom; Cat. No. ab189494) overnight followed by the secondary antibody AF488 plus-conjugated anti-rabbit (1:350; ThermoFisher) for 1 h at RT, shaking. Both antibodies were diluted in InsidePerm solution (Miltenyi; Cat. No. 130-090-477). Slides were mounted with DAPI-containing mounting solution (Merck) and digital captions were generated with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss). Exposure was optimized independently for each image and no quantification of brightness was carried out. The number of immunopositive cells with nuclear SIRT1 was determined by cell counting in at least five randomly selected fields/well using ImageJ software.