FIG 3.

The E2 protein interacts with mitochondrial proteins. (A) Tandem affinity purification (TAP) assay of the E2 protein. 293T cells were transfected with pCTAP-E2 (TAP-E2) or the control empty plasmid (Ctrl) and processed for TAP assays. After SDS-PAGE, the gel was stained by silver staining, and the specific bands were evaluated by mass spectrometry. (B) TAP detection of proteins of interest was confirmed by Western blotting with specific antibodies against each protein. (C) Plasmids expressing E2 and hemagglutinin (HA)-tagged interacting proteins were cotransfected into 293T cells, and localizations were detected using anti-E2 and anti-HA antibodies. Bars, 10 μm. (D) Pulldown assay of E2-interacting proteins. 293T cells were cotransfected with plasmids expressing E2 and HA-tagged E2-interacting proteins. Immunoprecipitation was conducted using an anti-HA antibody and detected with anti-HA and E2 antibodies. (E) Exogenous expression of the E2 protein. 293T cells were transfected with the HA-tagged HAX-1 plasmid, and 24 h after transfection, immunoprecipitation was performed with an anti-HA antibody. An arrowhead indicates full-length endogenous E2 protein detected with the anti-E2 antibody.