Repression mediated by dGW182 and its sufficiency domains appears to be independent of endogenous dGW182 and Argonaute 1. (A) S2 cells were treated with dsRNAs indicated above the lanes (dGW182, Ago1, or GFP as a negative control), and efficiencies of endogenous dGW182 and Ago1 depletions were analyzed by Western blotting with antibodies shown on the left. Expression of tubulin was estimated as a loading control. (B) dGW182 and its effector domains were transfected in S2 cells depleted of endogenous dGW182 or Ago1 and, as negative controls, in GFP-dsRNA-treated or untreated cells. Transfections were largely performed and analyzed as in Figure 1. In tethering experiments, firefly luciferase activity was expressed as a percentage of that in the presence of NHA-lacZ, for each RNAi depletion. To additionally control for efficient depletion of endogenous Ago1 and dGW182, firefly luciferase reporter containing miRNA-9b target sites (FLuc-nerfin) (Behm-Ansmant et al. 2006) was co-transfected with either miRNA-9b-encoding plasmid or the empty vector. As in all transfection experiments, RLuc was coexpressed as a transfection control. Firefly luciferase activity was normalized to that of Renilla luciferase and presented as a percentage of FLuc-nerfin expression in the presence of the empty vector.
