Figure 3.

Properties of arginine methylated E2F-1. (A) Levels of WT E2F-1, KK and KKK after transfection of the indicated expression vectors (1 μg) into U2OS cells and immunoblotting with HA11; – indicates untransfected cells. (B) Stability of WT E2F-1, and the KK and KKK mutants. Expression vectors (1 μg) encoding WT E2F-1, KK and KKK mutants were transfected into U2OS cells for 48 h. Cells were treated with 100 μg/ml of cyclohexamide and then harvested at 0, 2, 4, 6 h post-treatment time points as indicated for subsequent immunoblotting (i), and further quantitated (ii). The HA11 antibody was used for immunoblotting and actin served as protein loading control; n=2. Both the KKK (red) and KK (yellow) mutants had similarly increased half-life compared with WT (blue) E2F-1 (from 75 to 25 min, respectively). (C) Ubiquitination of WT E2F-1 and the KKK mutant. Cells were transiently transfected with expression vectors encoding WT E2F-1 or KKK (2 μg), together with His₆-ubiquitin (4 μg) as indicated, and treated with MG132 (20 nM) for 4 h before harvesting. Cell lysates and Ni²⁺ pull-down eluates were analysed as described. (D) Effect of PRMT5 on E2F-1 ubiquitination: U2OS cells were transfected with PRMT5 (P) or control (C) siRNA. After 24 h, cells were transfected with expression vectors encoding WT E2F-1 or the KKK mutant (2 μg) and His₆-ubiquitin (4 μg) as indicated. Cells were treated with MG132 (20 nM) for 4 h before being collected. Cell lysates and Ni²⁺ pull-down eluates were analysed as described. Graphical presentation of quantitation of ubiquitin signals was performed using ImageJ 1.43u, and the input protein levels are shown underneath. (E) PRMT5 siRNA increases E2F-1 protein levels. PRMT5 (P) or control (C) non-targeting siRNA was transfected into U2OS cells, and cells harvested 72 h post-transfection with or without etoposide (Et) treatment (10 μM) in the last 16 h. Extracts were immunoblotted with anti-PRMT5 and E2F-1, and GAPDH levels served as a loading control. (F) Protein levels of E2F-1 target genes in PRMT5 siRNA-treated cells. PRMT5 (P) or control (C) non-targeting siRNA was transfected into U2OS cells and cells were harvested 72 h post-transfection with or without etoposide (Et; 10 μM) or doxorubicin (Dox; 2 μM) treatment in the last 16 h. Extracts were immunoblotted with anti-PRMT5, E2F-1, p73, Chk2 and Chk1 as indicated. Levels of GAPDH served as the loading control.