Tat co-immunoprecipitation with Dicer requires RNA. A) 293T cells were transfected with pcDNA-Dicer-myc (lane 1) or cotransfected with pcDNA-Dicer-myc and pcDNA-wtTat-flag (lane 2) or Tat point mutants, TatK41A or TatK51A (lane 3 and 4). 48 hours later, cell lysates were immunoprecipitated with anti-myc beads overnight at 4°C. Dicer-immunoprecipitates were assessed by Western blotting using anti-myc (top panel) and co-immunoprecipitated Tat was detected using anti-flag (middle panel). As a control, the amounts of wt Tat and Tat mutants were verified in total cell lysates (lower panel). B) Co-immunoprecipitation analyses of transfected samples after no treatment (lane 1 to 4) or treatment with 50 μg/ml of RNase A (lanes 5 to 8). In addition to immunoblotting for Dicer and Tat, presence of TRBP in the immunoprecipitations was also analyzed.