Figure 1: POH1 is a positive regulator of E2F1 stability.

(a) For each DUB protein indicated, a pool of three independent siRNAs was transfected into PLC/PRF/5 cells. The protein levels of E2F1 were analysed by immunoblotting and normalized to that of the control GAPDH. The relative expression compared with that of the siRNA control is presented in rank order. (b,c) Immunoblot analysis of POH1 and E2F1 in human liver tumour cells (b) and mouse LPCs (c) transfected with the indicated siRNAs. Untransfected cells were shown as parental controls. (d) Immunoblot analysis of POH1 and E2F1 in human liver tumour cells with or without POH1-Flag expression. (e) Luciferase assay in HEK293T cells co-transfected with the reporter plasmids combined with the indicated siRNAs (left panel) or with POH1 and the vector control, respectively (right panel). Data are mean±s.d. (by t-test analysis, P values are shown in the graph, n=3). (f) SMMC-7721 cells transfected with control siRNA or POH1 siRNA were cultured for 72 h. Cell lysates were immunoprecipitated with anti-E2F1 antibody, and the immunocomplexes were immunoblotted with antibodies against UB and E2F1. (g) Human liver tumour cells transfected with POH1-Flag were immunoprecipitated with Flag-M2 agarose beads. The eluates were immunoblotted for the detection of E2F1. (h) HEK293T cells were co-transfected with E2F1-His and POH1-Flag followed by immunoprecipitation using anti-His antibody or IgG. The immunocomplexes were analysed by immunoblotting. (i) Endogenous E2F1 proteins were immunoprecipitated with anti-E2F1 antibody or IgG, and then analysed by immunoblotting. (j) PLC/PRF/5 cells with or without stable expression of POH1-Flag were treated with CHX (100 μg ml⁻¹) for the indicated time points. The cell lysates were examined by immunoblotting (left panel). A plot of normalized amount of E2F1 protein is shown (right panel). (k) SMMC-7721 cells were transfected with either control siRNA or POH1 siRNA for 48 h, followed by CHX(100 μg ml⁻¹) treatment for the indicated times. The cell extracts were analysed by immunoblotting (left panel). A plot of normalized amount of E2F1 protein is shown (right panel). Data in panels (j,k) are mean±s.d. (by ANOVA analysis, P values are shown in the graphs, n=3).