FIG 5.

Mode of interaction of ENY2 with E1A. (A) Effect of TRRAP depletion. HeLa cells were transfected with siRNAs directed against TRRAP or control siRNA, the WCLs were immunoprecipitated with the E1A Ab (M73), and the Western blots were probed with Abs against TRRAP, USP22, ATXN7L3, ENY2, and E1A (M73). (B) GST pulldown assay for E1A-interacting proteins. The WCLs from HeLa cells were incubated with GST or GST-E1A recombinant proteins, and the interacting proteins were purified by using the glutathione-agarose affinity beads. The proteins bound to the beads were analyzed in the flowthrough fractions by Western blotting using Abs against various E1A-interacting proteins. (C) Competitive interaction of ENY2 and TRRAP. HeLa cells were transfected with E1A 12S wt (pLPC-12S) with or without TRRAP and two different concentrations of ENY2. The interactions of TRRAP, ENY2, and pRb with E1A were analyzed by immunoprecipitation with the E1A Ab (M73) and Western blotting.