FIG. 5.

Mapping of the interaction domain of Agno protein with T antigen. Whole-cell extracts from HJC-15b cells were incubated with either GST alone (lane 2), GST-Agno (lane 3), or deletion mutants of GST-Agno fusion proteins (lanes 4 to 9) immobilized on glutathione-Sepharose beads. Bound complexes were washed extensively, resolved by SDS-PAGE, and analyzed by Western blotting using an anti-T-antigen antibody (Ab-2 416). HJC-15b whole-cell extract was loaded as a migration control (lane 1). The bracket indicates the position of bound T antigen. (B) Analysis of the GST and GST-Agno protein and its deletion mutants by SDS-PAGE. The similarly sized nonspecific bacterial protein which was copurified in our protein purification assays is indicated by an arrowhead. (C) Summary of the results obtained from in vitro mapping assays. The ability of Agno protein and its deletion mutants to interact with T antigen is depicted on the right as follows: ++++ or +++, very strong interaction; ++, strong interaction; +, reduced interaction; −, no interaction.