GW182 can associate with multiple Ago2 proteins. FLAG/SBP-Ago2 and HA-Ago2 were coexpressed with or without FLAG-GW182 1–910 or FLAG-GW182 1–910 (W470A and W755A) in HEK293F cells. (A) SBP pull-down assays were performed, and the precipitates were analyzed by Western blotting using an anti-FLAG M2 antibody (top), an anti-HA antibody (middle), and an anti-hAgo2 antibody (bottom). (Lane L) Cell lysate (1.25 μL equivalent); (lane E) fraction eluted with biotin (300 μL of cell lysate was subjected to the assay and 14% of the eluted fraction was loaded). (B) HA-Ago2 protein was precipitated with anti-HA agarose beads, and the precipitates were analyzed by an anti-FLAG M2 (top), an anti-HA (middle), and an anti-hAgo2 (bottom) Western blotting. (Lane L) Cell lysate (1.25 μL equivalent); (lane E) fraction eluted with 100 μg/mL of HA peptide solution. (300 μL of cell lysate was subjected to the assay and 14% of the eluted fraction was loaded).
