P16ink4a

Briefly, EPCs were washed 3 times with ice-cold phosphate-buffered saline (PBS), then lysed in RIPA lysate (Applygen Technologies Inc, Beijing, China) for 30 min on ice. The solution of EPCs was centrifuged at 4°C and 12000×g for 5 min. BCA protein quantitation kit (Wellbio Inc, Changsha, China) was used for protein measurement. Protein was mixed 1: 1 with 2×SDS loading buffer (20% glycerol, 4% sodium dodecyl sulfate, 3.12% dithiothreitol, 0.2% bromphenol blue, and 0.1 mol/l Tris HCl, pH 6.8, all from Sigma, Santa Clara, CA, USA), and incubated at 100°C for 4 min. Equal amounts of protein for each sample were separated by 10–12% SDS-polyacrylamide gel run at 120V for 90 min and blotted onto a polyvinylidene difluoride microporous membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight with primary antibody (eNOS: 1: 1000, Proteintech, Chicago, IL, USA; p16(INK4a): 1: 200, Santa Cruz Biotechnology, Santa Cruz, TX, USA), and subsequently washed 3 times with Tris-buffered-saline with Tween (TBS-T) and visualized following incubation with horseradish peroxidase-conjugated secondary antibody (1: 3000) for 1 h, followed by washing with TBS-T again. Immunoreactive bands were developed using an enhanced chemiluminescent substrate (Thermo, Waltham, MA, USA). After exposure, the films were scanned and analyzed using Quantity One (Bio-Rad, CA, USA).