Figure 4

APH-2 interacts with Tax2B and inhibits Tax2B-mediated activation of AP-1. (A and B) APH-2 neutralizes the stimulatory effect of Tax2B on AP-1 activity. 293T cells were transiently co-transfected with the pAP-1 reporter vector as well as the indicated expression plasmids. Two days after transfection, the cells were lysed and luciferase activities were measured. The graphs show an average from at least three independent experiments. The proteins levels were analysed by Western blot using the indicated antibodies. (C) APH-2 interacts with Tax2B in vitro. Purified Tax2B-His was incubated with GST or GST-APH-2 and a pull-down was carried out. The precipitate was analyzed using His antibodies (left panel). The purified proteins were visualized by Coomassie blue staining (right panel). (D) APH-2 forms a complex with Tax2B in vivo. 293T cells were transiently transfected with the indicated expression plasmids. Nuclear extracts were prepared two days after transfection and subjected to immunoprecipitation with His antibodies. Proteins in the immunoprecipitates were analyzed by Western blot using the indicated antibodies (WB).