Fig. 5. Differential binding of Tat-wt and ARM mutants to the PCAF BD and to TAR RNA. (A) HA-PCAF and Tat-FLAG expression vectors (wild-type or mutant proteins) were transfected in HEK 293 cells. In the two upper panels, cellular lysates were directly assayed by western blotting (WB) with the indicated monoclonal antibodies (αHA for PCAF and αFLAG for Tat) to assess the level of protein expression after transfection (Pre-IP Lysate). In the lower two panels, cellular lysates were subjected to Tat immunoprecipitation with anti-FLAG antiserum, and the immunoprecipitated material was analyzed by western blot (WB) with the indicated monoclonal antibodies. (B) Binding of Tat-wt and ARM mutants to TAR. Tat and mutant Tat proteins were translated and biolabeled in vitro, incubated with a synthetic RNA corresponding to nucleotides 1–57 of TAR-biotin. After incubation, TAR and bound proteins was bound to streptavidin–agarose beads, centrifuged and washed. The bound proteins were eluted and analyzed by SDS–PAGE and autoradiography.