Rabbit anti sirt1

Western blot analysis and immunohistochemistry have been carried out as described previously^{33 (link)}. Antibodies for research were below: rabbit anti-PKM2, rabbit anti-Beclin1, rabbit anti-LC3, rabbit anti-p-AKT, and mouse/rabbit secondary antibody were purchased from Cell Signaling Technology; rabbit anti-AMPKα1, rabbit anti-p-AMPKα1, rabbit anti-SIRT1, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology. Immunoblotting of LC3 usually gives two bands: LC3-I and LC3-II. The LC3-II/LC3-I ratio correlates with the number of autophagosomes and is applied as the marker for autophagic maker^{34 (link)}. For immunohistochemistry analysis, we considered PKM2-positive expression when cases with more than 30% cancer cells were stained.

For western blotting, 20–40 μg of total protein from each sample was subjected to SDS-PAGE under reducing conditions. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies. Primary antibodies used were rabbit anti-COX-2 (Santa Cruz), rabbit anti-iNOS (Santa Cruz), rabbit anti-acetyl-p65 (Cell Signalling), rabbit anti-Total-p65 (Cell Signalling), rabbit anti-MAP2(Cell Signalling), rabbit anti-Nrf2 (Santa Cruz), rabbit anti-HO1 (Santa Cruz), rabbit anti-NQO1 (Santa Cruz), rabbit anti-SIRT1 (Santa Cruz) and rabbit anti-actin (Sigma). Primary antibodies were diluted in Tris-buffered saline (TBS), containing 0.1% Tween 20 (TBS-T) and 1 or 5% BSA. Membranes were incubated with the primary antibody overnight at 4 °C. After extensive washing (three times for 15 min each in TBS-T), proteins were detected by incubation with Alexa Fluor 680 goat anti-rabbit secondary antibody (1:10,000; Life Technologies) at room temperature for 1 h. Detection was done using a LICOR Odyssey Imager. All western blot experiments were carried out at least three times.

Western blots were performed using rabbit anti-SIRT1 (Santa Cruz, CA, USA), anti-PI3K(Abcam), anti-Casp3 (CST, MA, USA), and anti-TNF-α (CST) antibodies as well as a β-actin antibody (Abcam). The hippocampi were homogenized in ice-cold lysis buffer, and the protein concentrations were determined using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL) following centrifugation. The proteins were separated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)-ready gels (Bio-Rad, Hercules, CA) and then electro-transferred onto nitrocellulose membranes. After blocking the membranes with 5% milk in Tris-buffered saline (TBS) for 1 h, we exposed the bound proteins to antibodies overnight at 4 °C. Following extensive washing with TBST, a 1:2,000 dilution of goat anti-rabbit horseradish peroxidase (HRP) secondary antibody (Cell Signaling Technology) was applied, and the membrane was incubated for 1 h at room temperature. Following extensive washing with TBST, the blots were examined using Western Lightning ECL (Perkin Elmer Life Sciences), with β-actin as a normalization control. The protein band intensities were determined by densitometric analysis using the Image J software.