Sirt1 antibody

Sirt1 over-expressing and control C3HT101/2 cells were lysed in lysis buffer (50mM TRIS-HCl, pH 7.4/1% NP-40, 0.25% Sodium deoxycholate/150 mM NaCl, 1mM EDTA/1mM PMSF supplemented with protease inhibitor and phosphatase inhibitor). Marrow from tibiae and femora was flushed with the same buffer, then cleared by centrifugation at 12,000 RPM for 15 min at 4°C. Immunoprecipitation was carried out by preclearing 1 μg of protein in 300 μl lysis buffer with 30 μl protein A beads (Millipore) for 1 h and incubating the lysates with 4 μl of the SIRT1 antibody (Millipore, 07-131), rotating over night at 4°C. For precipitation, 30 μl protein A agarose beads were added followed by incubation at 4°C for 3 h. Immunoprecipitates were washed extensively and eluted twice with x2 Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore).

The antibodies against AMPKα1/2 (H-300), AMPKα1/2 (Thr172), Akt1 (B-1), CYT C (H-104), PGC-1 (H-300), p-Akt1/2/3 (Ser473), and p-MFN2 (H-68) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). The SIRT1 antibody was purchased from Milipore (Temecula, California, USA). The eNOS antibody was purchased from Assay Biotechnology Co. Ltd. (Sunnyvale, CA, USA). The p-eNOS (Ser1177) antibody was purchased from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). The blotting experiments were performed obeying to the manufacturer’s instruction manuals. The gray scale values from blotted proteins were measured using a scanning instrument, and the raw data of gray scale values were normalized by a gray scale value of the reference protein GAPDH available from each group.

Western blot analysis was conducted as previously reported [41 (link)]. Aortas were homogenized in ice cooled lysis buffer (1% Triton X-100, 50 mM Hepes [pH 7.4], 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, and protease inhibitor cocktail), followed by centrifugation at 15,000 rpm for 30 min at 4 °C and collection of supernatants (n = 5 for each group). Samples (2 to 10 μg) were resolved by 10% or 12.5% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Thereafter, the membrane was blotted with 5% skim milk for 1 h, reacted with various primary antibodies at 4 °C overnight, and reacted with secondary antibody on the following day. The samples were then visualized using Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific Inc., Waltham, MA, USA). Images were obtained by the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA) and quantified by PDQuest software (Bio-Rad, Hercules, CA, USA). For the primary antibody, anti-HO-1 antibody (Stress Gen Biotechnologies Inc., Victoria, BC, Canada), Sirt1 antibody (Merck Millipore, Billerica, MA, USA), and β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and for the secondary antibody, anti-rabbit IgG, HRP-linked whole antibody donkey (GE Healthcare, Buckinghamshire, UK) was used.