Figure 5

HIV-1 ARM confers interaction ability with components of RISC Loading Complex (RLC). HEK 293 T cells were co-transfected with His TRBP, His PACT and Myc Tat, Myc Rev, and their corresponding ARM deletion mutants in the presence and absence of Adox. 48 hours later cell lysates were subjected to co-immunoprecipitation as described in methods. TRBP and PACT immunoprecipitates were assessed by western blotting using anti-His and co-immunoprecipitated Tat, Rev and their ARM deletion mutants were detected using anti-myc (A and B). (C) and (D) show the relative binding of ARM deleted mutants with respect to wild type proteins with TRBP and PACT respectively. (E) shows co-immunoprecipitation of His-TRBP and HA-PACT with GST and GST fused HIV-1 ARM (TmGST and RmGST) using a similar protocol as in 5A (using Ni–NTA in first case and Glutathione Beads in second). (F) is co-immunoprecipitation analysis of His TRBP and His PACT with Myc Rev and RmGST in the presence and absence of 50 μg/ml of RNase A.