Figure 4

Identification of the BHEXIM1 domain responsible for binding to B-cyclin T1 and inhibiting BTat transactivation. (A) Schematic representation of HA-tagged truncation forms of BHEXIM1. (B) 293 T cells were co-transfected with an empty vector, BHEXIM1, or the indicated truncations together with Flag-B-cyclin T1. BHEXIM1 was immunoprecipitated with rabbit anti-HA agarose beads, and the immunoprecipitates were detected by western blot using a mouse anti-Flag (top) or mouse anti-HA (middle) antibody. The amount of B-cyclin T1 in the cell lysates was checked using an anti-Flag antibody (bottom). (C) Cf2Th cells were co-transfected with a BIV LTR-Luc plasmid (0.05 μg), pCMV-β-gal plasmid (0.1 μg), wild-type BHEXIM1 or the deletion form of BHEXIM1 (0.1 μg) with or without BTat (0.025 μg), and an empty vector to ensure that the total DNA concentration in each transfection mixture was constant. After 48 h, the relative luciferase activities were analyzed. All of the luciferase activities with Tat were divided by the luciferase activities without Tat. Error bars represent the SD of the means from three independent experiments.