Both GW182 fragments GW1Δ1 and GW1Δ10 coprecipitated with other human Ago proteins. (A) Ago 1 and Ago4, but not Ago3 mutant, coprecipitated with GW182 fragments GW1Δ1 and GW1Δ10. GFP-tagged Ago1, Ago3m (Ago3 mutant), or Ago4 was cotransfected with GST-GW1Δ10 (lanes 1–6) or GST-GW1Δ1 (lanes 7–12). Ago3m is missing an exon (“aa757-823”), the C-terminal 66 amino acids of the PIWI domain compared with the reference sequence (NM_024852.2). Both Ago1 (lanes 4,10) and Ago4 (lanes 6,12) were pulled down by GST-GW1Δ10 or GST-GW1Δ1. In comparison, Ago3m was absent from either pull-down (lanes 5,11). (B) Ago3 coprecipitated with GW182 fragment GW1Δ1. Flag-Ago3 (lanes 1,3) or Flag-Ago1 (lanes 2,4) was cotransfected with GST-GW1Δ1 (lanes 1–4). GFP–Ago3m (lanes 5,6) was cotransfected with GST-GW1Δ1 as a negative control.
