Anti klf2

Total protein was isolated from cells using cold radio immunoprecipitation assay buffer containing a protease inhibitor cocktail (Roche Applied Science, Penzberg, Bavaria, Germany). A total of 50 μg of protein was subjected to SDS-PAGE, and the blots were transferred onto a polyvinylidene difluoride membrane. After blocking, the membranes were incubated with anti-SIRT1 (Millipore, Temecula, CA, USA), anti-SIRT2 (Cell Signaling Technology, Danvers, MA, USA), anti-SIRT3 (Cell Signaling Technology), anti-SIRT5 (Millipore), anti-SIRT6 (Cell Signaling Technology), anti-eNOS (Cell Signaling Technology), anti-KLF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FOXM1 (Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology), and anti-FLAG (Sigma-Aldrich) antibodies. Antibodies against cell cycle regulators and checkpoint molecules, including cyclin D1, cyclin D3, p18 INK4C, p21 Waf1/Cip1, p27 Kip1, CDK2, CDK6, phospho-RB, and phospho-p53 (Ser15), were purchased from Cell Signaling Technology. Immune-reactive protein bands were visualized by chemiluminescence using ECL reagents (GE Healthcare, Fairfield, CT, USA). Protein expression was imaged in a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).