P18 ink4c

Western blotting (WB) was performed as described previously [22 (link)]. Briefly, total protein was extracted from the cells. Aliquots of total protein (10 μg) were electrophoresed on sodium dodecyl sulfate–polyacrylamide and 12% Tris–HCL gels (Bio-Rad Laboratories, CA, USA). The separated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) and incubated with primary antibodies overnight at 4 °C. The membranes were probed with the following antibodies: cyclin D1, p27 Kip1, CDK2, p18 INK 4C, CDK6, cyclin D3, p21 Waf1/Cip1, CDK4, pIκBα, IκBα, pro caspase 3, Ikaros, CK2α, c-Myc (1:1000 dilution; Cell Signaling Technology, Inc., MA, USA), and anti-β actin (1:3000 dilution; Sigma, Tokyo, Japan). Bound primary antibodies were detected using secondary antibodies (Cell Signaling Technology, Inc.) in a 1:10,000 dilution for anti-β actin and 1:2000 dilution for the other antibodies.

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).

Total cell lysates were prepared using RIPA buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS). Each protein sample (20–30 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). Antibodies against PAK2, Cyclin D3, CDK2, CDK4, CDK6, Cyclin D1, p18INK4C, p21Wafl/Cip1, p27Kip1, p53, Caspase 7, PARP, survivin, XIAP, Livin, Bad, phospho-Bad (S112), phospho-Bad (S136), and GAPDH were purchase from Cell Signaling Technology (Beverly, MA). Anti-Mcl-1, anti-Bax, HRP-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin was purchased from Sigma. The labeled proteins were visualized with Immobilon Western Chemiluminesent HRP Substrate kit (Millipore) or Power Opti-ECL Western blotting Detection reagent (Bionote, Hwaseong, Korea) and the images were captured by ImageQuant LAS 4000 (GE healthcare, Buckinghamshire, UK). The protein band intensity on western blots was quantified and normalized to that of β-actin by the densitometry analysis using ImageJ software (http://rsb.info.nih.gov/ij/).