Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression

Multiple Ago2-binding sites are present in the amino-terminal half of GW182. (A) Schematic representation of the GW182 fragments used in the SBP pull-down assay. The polypeptides that can bind Ago2 are represented by the white bars, while the black bars represent those that cannot bind Ago2. (B) Expression of Ago2 and each GW182 fragment in HEK293F cells. The expressed proteins were detected by Western blotting with an anti-FLAG M2 antibody. (C–E) SBP pull-down assays of Ago2 and GW182. HEK293F cells were cotransfected with FLAG-Ago2 and FLAG/SBP-GW182 expression vectors. SBP-pull down assays and Western blotting were performed as shown in Figure 1C.