Figure 4

cIAP1 is required for DNA damage-induced stabilization of E2F1. (a) Western blot analysis of E2F1 and cIAP1 in U2OS cells transfected with control or cIAP1-siRNA and treated with indicated concentration of etoposide for 6 h. β-Actin was used as loading control. (b) Ubiquitination profile of E2F1 in HeLa cells transfected with cIAP1 siRNA or cIAP1-encoding construct and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10μM etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes and the efficiency of siRNA were checked by a western blot analysis (input). β-Actin was used as loading control. (c) Ubiquitination assay performed in HeLa cells transfected with control or cIAP1 siRNA and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10μM etoposide. Ubiquitinated proteins were pulled-down by using K63 specific TUBEs and ubiquitinated E2F1 is revealed by using E2F1 antibody. (d) Western blot analysis of E2F1 in U2OS cells transfected with 3HA-E2F1 or 3HA-E2F1-K161/164R mutant and treated with indicated concentration of etoposide for 6 h. β-Actin was used as loading control. (e) Ubiquitination of E2F1 in HeLa cells transfected with pCMV-3HA-E2F1 or pCMV-3HA-E2F1 K161/164R, and with His-tagged ubiquitin wt, and then treated for 6 h with 10 μM etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both untreated samples. (f) U2OS cells were transfected with cIAP1 siRNA or treated with 17 nM GDC-0152 for 1 h, and then treated with 10 μM etoposide for 48 h. tp73 mRNA expression was measured by RT-qPCR. UT: untreated cells. Results were normalized to hprt mRNA and expressed relatively to control untreated cells. Mean±S.D. of three independent experiments