Figure 4

Interaction of jTat with CycT1 in vitro and in vivo. (A) Interaction of hTat and jTat with mammalian CycT1s. GST and GST-tagged proteins were immobilized on beads and incubated with the cell lysates as described in Methods. The pull-down complexes and 5% of cell lysate input were analyzed by western-blotting using anti-Flag antibody. The coomassie blue staining shows 10% of the amounts of the purified proteins utilized in this experiment. Numbers mark the molecular weight standards (MW). (B) Schematic representation of mammalian two-hybrid constructs. jAD; jTat residues 1-67. JH; jTat 1-67 fused to hTat 48-72. (C) HeLa cells were co-transfected with 500 ng J-NFκB or H-NFκB, 500 ng of the indicated Gal4 BD plasmid and 250 ng pFR-luc. Fold-induction shows the relative activity of pFR-luc reporter and reflects binding affinity between Tat and its cofactor. (D) HIV LTR activation in 3T3 cells by indicated Tats in the absence or presence of hCycT1. The amount of transfected pCMV-Tag2B-hCycT1 was 50 ng. T1; Cyclin T1 residues 1-272.