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. Author manuscript; available in PMC: 2019 Dec 6.
Published in final edited form as: Mol Cell. 2018 Nov 8;72(5):823–835.e5. doi: 10.1016/j.molcel.2018.09.019

Figure 2: HPV E6 promotes Cdt2 and USP46 interaction.

Figure 2:

(A) Rescue of Cdt2 stabilization after siUSP46 using siRNA-resistant USP46 overexpression. HeLa cells were transfected with indicated plasmids and siRNAs as indicated and immunoblotted. Lane 3: siRNA-resistant HisUSP46. (B) HeLa or U2OS cell lysates immuno-precipitated with control IgG or anti-Cdt2 antibodies, and immunoblotted. (C) E6-USP46-Cdt2 interaction reconstituted in vitro. HisUSP46 from 293T cell lysates bound to nickel-NTA beads in the presence of bacterially purified GST or GST-E6. Immunoblot of bound proteins shows cellular Cdt2 associating with column along with GST-E6. Right hand panel shows 10% input cell lysate or GST protein. (D) Bacterially purified GST or GST-E6 incubated with bacterially purified His-USP46, loaded onto a nickel-NTA column, washed, eluted and immunoblotted with indicated antibodies. (E) Bacterially purified E6 interacts with USP46 purified from mammalian cells. Left: Glutathione beads coated with GST or GST-16E6 were incubated with purified His-USP46 (lanes 1 and 3). Nickel beads coated with nothing or His-USP46 were incubated with GST-16E6 (lanes 2 and 4). Beads were washed and boiled in Laemmli buffer for immunoblotting. Right, Lanes 1 and 2: input (10%) purified GST-E6 or His-USP46. Lane 3: 293T cell lysate from which the HisUSP46 was purified to show that cellular Cdt2 has been removed from the purified His-USP46 in lane 2. (F) E6 promotes interaction between Cdt2 and USP46 but USP46 is essential for the interaction between E6 and Cdt2. HEK293T cells transiently transfected with plasmids or siRNAs indicated at top. 48 hr later, cell lysates were immunoprecipitated with anti-flag antibody and immunoblotted. Input: 15% of input lysates.