Figure 3: POH1 reverses K63- and K11-linked polyubiquitination of E2F1.
From: POH1 deubiquitylates and stabilizes E2F1 to promote tumour formation
(a) HEK293T cells overexpressing E2F1-His, POH1-Flag, and HA-tagged, different forms of ubiquitin were treated with MG132 (10 μM) for 6 h. Cell lysates were immunoprecipitated using anti-His antibody. Ubiquitination levels were detected using anti-HA antibody. (b) SMMC-7721 cells transfected with UB(WT) or UB(K63R) were cultured for 72 h in the presence of control siRNA or POH1 siRNA. Cell lysates were analysed by immunoblotting using the indicated antibodies. (c) A schematic diagram of the JAMM motif within the MPN domain of POH1. (d) HEK293T cells were co-transfected with E2F1-His, vector, or the POH1-Flag, and UB (K63)-HA constructs. Cell lysates were immunoprecipitated with anti-His antibody and immunoblotted with the indicated antibodies. (e) UB (K63)-HA modified E2F1-His proteins were purified from HEK293T cells with protein G beads and anti-His antibody. The protein complexes containing POH1-Flag or the Flag-tagged C120S or H113Q mutants of POH1 were purified from HEK293T cells with anti-Flag M2 beads. The purified UB (K63)-HA-E2F1 and POH1-Flag protein complexes were incubated for 1 h in the reaction buffer. After the reaction, E2F1-His proteins were further purified with anti-His antibody and immunoblotted with antibodies against HA and His. (f) SMMC-7721 cells were transfected with control siRNA or POH1 siRNA together with the siRNA-resistant (siR-R) forms of Flag-tagged WT or mutant POH1. The cells were collected 72 h after transfection and immunoblotted with the indicated antibodies. (g) SMMC-7721 cells stably expressing the siRNA-resistant POH1 (C120S)-Flag were transfected with control siRNA or POH1 siRNA and cultured for 72 h.The cell lysates were analysed by immunoblotting to determine the levels of E2F1 and total ubiquitin-modified proteins.
