FIGURE 5.

AGO1 mutants impaired in GW182-binding are also impaired in the complementation assay. (A–D) S2 cells were transfected with (Control) GFP-siRNA or (AGO1 knockdown) AGO1-siRNA plus a mixture of three plasmids: one expressing the indicated F-Luc reporters; another expressing miRNA primary transcripts (+miRNA) or the corresponding empty vector (−); and a third expressing Renilla luciferase (R-Luc). Plasmids encoding siRNA-resistant versions of wild-type or mutant HA-AGO1 proteins were included in the transfection mixtures, as indicated. Control cells were cotransfected with a plasmid encoding HA-MBP. Firefly luciferase activities were normalized to those of the Renilla luciferase transfection control and analyzed as described in Figure 2.