Figure 3.

NAMPT inhibition or KD results in increased p73 levels independent of p53. (a and b) Lysates from FK866-treated (a) or NAMPT KD (b) cells were subjected to western blot (WB) analysis for p73, ΔNp73, SIRT1, p53 and actin. (c) Relative expression levels of p73 mRNA from untreated (control) and FK866-treated U2OS cells were quantified by quantitative PCR (Q-PCR). Results represent three independent experiments. (di) Western blot analysis of overall acetylation (left panel) and ubiquitination (right panel) in FK866-treated U2OS cells. (ii and iii) Lysates from FK866-treated cells were subjected to immunoprecipitation (IP) using an anti-p73 antibody followed by western blot analysis for protein acetylation (ii) and ubiquitination (iii). (iv) Western blot showing the effect of MG132 treatment (1 μM for 24 h) on p73 levels in U2OS cells. (e) FK866 enhances p73 stability. U2OS cells were treated with FK866 for 48 h and then CHX (20 μM) was added. Cells were collected at different time points and analysed by immunoblotting to measure p73 protein levels. Wt, wild type; NS, nonsignificant