Anti sirt1 antibody

SIRT1 enzymatic activity was measured using a commercial kit (ab156065; Abcam). According to the manufacturer's directions, fresh intestinal tissues were immunoprecipitated (Immunoprecipitation Kit; Proteintech, Chicago, IL, USA) with anti-SIRT1 antibody (Santa Cruz Biotechnology). Then, the reaction mixture containing fluoro-substrate peptide solution and protein A agarose beads was added, and NAD-dependent deacetylase activity was measured based on fluorescence intensity at 1-2 min intervals at Ex/Em = 350/460 nm in SpectraMax M5. The activity is presented as the relative value compared to the control group.

Immunoprecipitation analysis was performed using standard protocols based on previously described methods22 (link). Cultured cells were lysed with Cell Lysis Buffer (Beyotime) supplemented with a protease inhibitor cocktail (Roche). A total of 40 μg protein were separated on a 12% SDS–polyacrylamide gel (SDS-PAGE). After electrophoresis, the proteins were transferred to PVDF membranes, followed by antigen-blocking in 5% fat-free milk. The membranes were then probed with the indicated antibodies overnight at 4 °C, and then washed and incubated with primary-antibody-matched and HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 hours. Finally, the membranes were washed and visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following primary antibodies were used: anti-Myc antibody (Santa Cruz Biotechnology), anti-GST antibody (Santa Cruz Biotechnology), anti-SIRT1 antibody (Santa Cruz Biotechnology), anti-Nkx2.5 antibody (Santa Cruz Biotechnology), anti-HA antibody (Santa Cruz Biotechnology), anti-Flag antibody (Sigma), anti-acetylated lysine (ac-K) antibody (Cell Signaling Technology), and anti-actin antibody (Santa Cruz Biotechnology).

Immunoprecipitation was performed as before with minor modifications [32 ]. 500 μg of total rMC lysate diluted in extraction buffer were immunoprecipitated with rabbit anti-SIRT1 (Santa Cruz) using Protein A/G agarose beads. The samples were subjected to Western blotting against nitrotyrosine (Upstate).
To determine the carbonylation of SIRT1, blots were probed first with anti-SIRT1 antibody (Santa Cruz). After stripping, the membranes were incubated with 20% methanol, 80% Tris-buffered saline for 5 min, then incubated with 0.5 mM 2,4-dinitrophenylhydrazine (DNP, Sigma) to detect carbonyl groups associated with aldehydes and ketones for 30 min at room temperature. The membranes were washed and then incubated overnight in anti-DNP antibody (Merck Millipore, Billerica, MA, USA), as previously described [33 (link)].

Optic nerve specimens (4-mm lengths) were gathered and homogenized in protein extraction buffer 1 week after injection. Homogenized samples were then centrifuged at 15,000×g for 15 min at 4 °C. Protein concentrations were determined with the supernatants. Each sample (3 μg) was applied and subjected to the mini gel (Bio-Rad Laboratories) and transferred to enhanced chemiluminescent membrane (EMD Millipore Corporation, Temecula, CA). The membranes were blocked with 5% skim milk with tris buffered saline (TBS) containing Tween-20 and reacted with anti-p62 antibody (MBL Life Science, Nagoya, Japan), anti-LC3 antibody (MBL Life Science), anti-SIRT1 antibody (Santa Cruz Biotechnology), anti-NRK1 antibody (Lifespan Biosciences Inc. Seattle, WA) or anti-β-actin antibody (Sigma-Aldrich). After three times washing, the membranes were reacted with anti-rabbit or anti-mouse peroxidase-labeled secondary antibody (MP Biochemicals, Solo, OH). Immunoblotting was visualized with a chemiluminescence detection system (ECL Plus Western Blotting Detection Reagents, Amersham Pharmacia Biotech).

Oocytes were collected and washed with PBS supplemented with 0.1% PVA and then fixed with 4% PFA at RT for 30 min. The oocytes were then permeabilized with 1% Triton-100 in PBS at RT for 8–12 h and blocked with 1% BSA for 1 h at RT. After washing with 0.05% Tween-20 containing 0.1% Triton-100 in PBS, the oocytes were incubated with anti-α-tubulin FITC antibodies (Thermo Fisher Scientific, USA) at a dilution of 1:200, and an anti-SIRT1 antibody (Santa Cruz, CA, USA) at a dilution of 1:200 overnight at 4 °C. After washing in 0.05% Tween-20 containing 0.1% Triton-100 in PBS, the oocytes were stained with Alexa Fluor 488 (ZSGB-BIO, Beijing, China) secondary antibody for 1 h at RT. Oocytes were incubated in 10 μg/ml propidium iodide (PI) at RT to view the nucleus. After several washes, the oocytes were mounted on glass slides, and images were captured under a confocal laser scanning microscope (LAS - Leica TCS-SP8, Germany) with the same scanning settings.