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. Author manuscript; available in PMC: 2015 Jan 31.
Published in final edited form as: Mol Cancer Res. 2014 Aug 20;13(1):41–49. doi: 10.1158/1541-7786.MCR-14-0239

Figure 2.

Figure 2

SIRT1 regulates p27kip1 stability through the ubuquitin-proteolysis pathway. A & B. SIRT1 silencing induces significant increase in p27kip1 protein half-life. SIRT1-silenced and shRNA-control H1299 (A) and H460 (B) cells were treated with cycloheximide (10 µg/ml) for 0, 5, 8, 25 hr. Cell extracts were made from the treated cells and immunoblot analysis was performed with p27 and β-actin antibodies (left panels). The p27 intensity was analyzed using image J software and the p27 half-lives were calculated as the time required for each p27 protein decrease to 50% of its initial level (right panels). C. Proteasome activity inhibition with MG132 blocks SIRT1 overexpression-mediated p27Kip1 downregulation. His tagged-SIRT1 expression plasmid and vector control plasmid were transfected into H1299 cells. After 48 hrs, the cells were further treated with cycloheximide (10µg/ml) together with MG132 (10uM) or vehicle for 4hrs. Immunoblot analysis was performed with p27, His-tag, and β-actin antibodies.