Western Blot Analysis of Cell Signaling

Western blot analysis was performed on the basis of a previous report [65 (link)]. Briefly, HPDL cells were lysed in RIPA buffer (Millipore, MA, USA) containing a protease inhibitor cocktail (Roche, IN, USA), 1 mM sodium orthovanadate (Sigma-Aldrich), 1 mM Sodium fluoride, and 10 mM β-glycerophosphoric acid (Wako). Protein concentrations of the lysates were quantified by the Bradford Assay (Bio-Rad, CA, USA). Lysates were denatured in 5× Laemmli buffer containing 2-mercaptoethanol by boiling for 10 min at 95°C. After cooling, the proteins were separated by SDS-PAGE under reducing conditions and transferred to a PVDF membrane (GE Healthcare, IN, USA). Membranes were blocked with 5% dry skim milk for 1 h and then probed with the following primary antibodies: mouse anti-human p53, rabbit anti-human p16, goat anti-human CTGF (Santa Cruz, TX, USA), rabbit anti-human p21, mouse anti-human Rb, rabbit anti-human SIRT1 (CST), and mouse anti-human β-actin (Sigma-Aldrich). After washing, membranes were incubated with secondary antibodies, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG or donkey anti-goat IgG (CST), and visualized with ECL prime Western Blotting Detection Reagents (GE Healthcare).