Anti α tublin

Samples of tissue or cell extract were separated by SDS–polyacrylamide gel electrophoresis, and the proteins were transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Bedford, MA, USA) in a Trans-Blot SD Cell (BIO-RAD, Hercules, CA, USA). The membrane was blocked with 0.5% TSA Blocking Reagent (PerkinElmer Life Sciences, Waltham, MA, USA), incubated overnight at 4 °C with one of the primary antibodies—anti-NAMPT (1:1000,#A300-372A; Bethyl Laboratories Inc, Montgomery, TX, USA), anti-SIRT1 (# 07-131; Millipore), anti-SIRT3 (#5490; Cell Signaling Technology), anti- β-actin (#4970; Cell Signaling Technology), anti-Cox IV (#ab16056;Abcam, Cambridge, UK), anti- LaminB1 (#ab16048; Abcam), anti-α-tublin (#T9026; Sigma-Aldrich, St. Louis, MO, USA), anti-acetyllysine (#9441; Cell Signaling Technology), anti-SOD2 (#ADI-SOD-111; Enzo life Sciences, NY, USA), and anti-acetylated SOD2^{19 (link)} (#ab214675; Abcam)—and then incubated with horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) for 1 h. The signals were detected using ECL Western Blotting Detection Reagents (GE healthcare Limited, Buckinghamshire, UK). Signal intensities were quantified using the ImageJ program and normalized to β-actin.