Horseradish peroxidase conjugated anti mouse or anti goat secondary antibody

For the validation of protein expression changes by immunoblotting, 20 mg of protein extract was separated on 8% and 12% SDS polyacrylamide gels according to Laemmli [34 (link)]. Proteins were transferred to nitrocellulose membranes (GE Healthcare) using a semidry blotting system at 100 mA for 90 min. Membranes were saturated for 1 h with 5% advance blocking reagent (GE Healthcare) in TBS (50 mM Tris.HCl, pH 7.6 and 150 mM NaCl) containing 0.1% Tween 20 (TBS/T).
Blots were incubated overnight at +4°C with antibodies against either SIRT1, SIRT3, acetyl-p53, phospho-p53 or p21 protein (Cell Signaling) with alpha-tubulin (Cell Signaling) as the loading control. After washing three times in TBS/T, blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-goat secondary antibody (Santa Cruz Biotechnology) in blocking buffer (TBS/T with 5% w/v advance blocking reagent). Immunodetection was performed with ECL advance Western blotting detection kit (GE Healthcare). The protein bands were quantified using ImageQuant 5.2 software (GE Healthcare) by integration of all pixel values in the band area after background correction, and normalized to the alpha-tubulin expression. The level of alpha-tubulin is not affected by irradiation in endothelial cells and was therefore used for normalization [25 (link)].