Goat anti rabbit horseradish peroxidase hrp secondary antibody

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.

Approximately, 20 mg of TA muscle was homogenised in 1x radioimmunoprecipitation assay (RIPA) lysis buffer containing 1x protease inhibitor cocktail (Sigma-Aldrich) and quantified by the BCA Protein Assay kit (Thermo Scientific). Alternatively, 10 μm thick EDL muscle cross sections were placed into 50 μl of 2x Laemmli sample buffer and underwent two freeze thaw cycles at −80°C. Proteins (7.5 μg of unfractionated TA homogenates or 15 μl of EDL cryosection lysates) were separated on an Any kD™ Mini-PROTEAN® TGX Stain-Free™ gel (BioRad). Gels were then activated and imaged using the ChemiDoc MP System (BioRad) as per manufacturer's instructions and then transferred onto a PVDF membrane. These were blocked with 5% skim milk/TBST, incubated with an anti:SEPS1 antibody (HPA010025; Sigma-Aldrich; diluted 1 : 200 in 1% skim milk/TBST), and followed by a goat anti-rabbit horse radish peroxidase (HRP) secondary antibody (Cell Signaling Technologies: diluted 1 : 5000). SEPS1 bands were detected with enhanced chemiluminescence and imaged using the ChemiDoc MP System. The optical density of the SEPS1 bands and the protein bands on the TGX Stain-Free gels were analysed using Image Lab™ software (Bio-Rad), and SEPS1 protein levels were normalised to the total optical density of all protein bands.