GW182 functions downstream from miRNA loading onto AGO1. (A) S2 cells were treated (Control) GFP or (GW182 KD) GW182 dsRNAs on days 0 and 4, and transfected on day 6 with plasmids expressing HA-MBP or HA-AGO1. Proteins were immunoprecipitated using a monoclonal anti-HA antibody. Inputs (1.5%) and immunoprecipitates (30%) were analyzed by Western blotting using a polyclonal anti-HA antibody. The association between HA-AGO1 and miRNAs was analyzed by Northern blotting. tRNAAla served as a loading control for the Northern blots. (B) The effectiveness of the GW182 depletion was analyzed by Western blotting using anti-GW182 antibodies. (Lanes 1–4) Dilutions of control cells (treated with GFP dsRNA) were loaded. Tubulin served as a loading control. (C) Lysates from S2 cells expressing HA-tagged versions of MBP, wild-type GW182, or GW182 mutants were immunoprecipitated using a monoclonal anti-HA antibody. Inputs (1.5%) and immunoprecipitates (30%) were analyzed by Northern blotting, as described in panel A. Endogenous AGO1 was detected by Western blotting using anti-AGO1 antibodies.
