Figure 6.

Overexpression of the NED fused to the AGO1-binding motif rescues RNAi knockdown of endogenous dGW182. (A) Endogenous dGW182 was depleted in Drosophila S2 cells with dsRNA (black bars); a batch of cells was treated with GFP-specific dsRNA as a negative control (open bars). Cells were transfected with firefly luciferase reporter containing miRNA-9b target sites (FLuc-nerfin) and with either the miRNA-9b-encoding plasmid or the empty vector. As in all transfection experiments, RLuc was co-expressed as a transfection control; expression levels of firefly luciferase were normalized to Renilla luciferase activity and expressed as a percentage of the firefly luciferase activity in the presence of the empty vector. To rescue the knockdown of endogenous dGW182, increasing amounts of plasmids encoding NHA-dGW182 or its N-terminal fragments were co-transfected: the NED fused to the AGO1-binding motif (1–490), either wild-type or non-functional point mutants (M2/4a/5 and 6W6A), and the 1–605 construct. Expression of NHA-dGW182 fragments was estimated by western blotting with anti-HA antibody. Increasing amounts of NHA-lacZ were co-transfected for a negative control. (B) Efficiency of the endogenous dGW182 knockdown was estimated by western blot analysis. S2 cells were treated with dsRNAs, indicated above the lanes (dGW182 or GFP as a negative control), and analyzed by western blotting with the antibodies shown on the left. Expression of tubulin was estimated as a loading control.