The amino-terminal half of GW182, containing GW/WG repeats, binds Ago2. (A) Schematic representation of the GW182 fragments used in the SBP pull-down assay. The polypeptides that bound Ago2 are represented by the white bars, while the black bars represent fragments that did not bind Ago2. (B) Expression of Ago2 and the GW182 fragments in HEK293F cells. The expressed proteins were detected by Western blotting with an anti-FLAG M2 antibody. The sizes of the molecular markers are indicated on the left (lane M). (Lanes F,FS) FLAG- and FLAG/SBP-tagged proteins, respectively. (C–F) SBP pull-down assay of Ago2 and GW182. HEK293F cells were cotransfected with FLAG/SBP or FLAG-tagged Ago2 and the GW182 expression vectors. After 48 h, the cells were harvested and the prepared cell lysates were subjected to SBP pull-down assays with (+) or without (−) RNaseA. The precipitates were analyzed by Western blotting using an anti-FLAG M2 antibody. The positions of the GW182 fragments are indicated with triangles on the right. (Lane L) The cell lysate sample.
