FIG. 4.

In vitro and in vivo interaction between Agno protein and T antigen. (A) Agno protein interacts with T antigen in GST pull-down assays. Whole-cell extracts prepared from HJC-15b cells, which express JCV T antigen constitutively, were incubated with either GST alone (lane 2) or GST-Agno protein (lane 3) as described in Materials and Methods. Beads were washed extensively and proteins interacting with GST or GST-T-Agno were resolved by SDS-PAGE and analyzed by Western blotting using anti-T-antigen antibody (Ab-2 416). (B) Agno coimmunoprecipitates with T antigen. Five hundred micrograms of whole-cell extracts prepared from HJC-15b cells and HJC-15b cells transfected with an Agno expression plasmid (pEBV-Agno) was immunoprecipitated either with normal serum (α-pre) (lane 4) or anti-T7 (α-T7) (lane 5) antibodies. Immunocomplexes were resolved on an SDS–10% PAGE and analyzed by Western blotting for the presence of T antigen with an anti-T-antigen antibody (Ab-2 416). Tfxn, transfection. The arrowhead indicates the position of immunoglobulin heavy chain detected by the secondary antibody. (C) Whole-cell extracts from HJC-15b cells treated with either ethidium bromide (100 ng/ml, lane 4) (Et-Br), DNase I (0.2 U/μg of protein, lane 5) (47), or RNaseI (0.5 U/μg of protein, lane 6). In lane 1 for each panel, the whole-cell extracts prepared from HJC-15b cells were loaded as a migration control for T antigen.