Figure 3.
PPD–Dicer interactions are direct. (A) Protein A–sepharose beads coated with affinity-purified Abs against Dicer or the control protein GAR1 were incubated with mixtures of purified Dicer and GST–AGO2 (upper panel), GST–Hiwi (middle panel) or GST alone (lower panel). Bead-associated (B) and unbound (S) material was subjected to SDS–PAGE and immunoblotting with anti-GST Abs. (B) Different combinations of pGBKT7 (AGO2 or Hiwi subdomains) and pGADT7 (Dicer subdomains) constructs were transformed into AH109 strain and tested for interaction by growth on selective media. Four serial dilutions were spotted for each combination. Scoring ranged from − to ++++ depending upon how well the strains grew on media lacking leucine, tryptophan, adenine and histidine. Graded examples of some PPD–Dicer interactions are shown. A complete listing of all the tested interactions is shown in supplementary table 2 online.