Fig 1: Dicer–TRBP induces cleavage in fully base-paired pre-miRNAs. (A) Schematic overview of the pull-down experiment for Dicer–TRBP complex isolation. (B) Western blotting analysis of the Dicer pull-down experiment. The indicated recombinant proteins were mixed and incubated for 30 min on ice, then incubated with beads bound with anti-TRBP specific antibody at 4°C overnight and eluted with SDS sample buffer. Eluted proteins (a half of the pull-down eluate volume) and recombinant Dicer (0.187 pmole), were analyzed by western blotting with an anti-Dicer specific antibody. (C) Dicer/TRBP cleavage assay of the fully base-paired pre-miRNAs followed by northern blotting. Recombinant Dicer (0.2U, 0.05 pmole) and reconstituted Dicer/TRBP complex (10% of the pull-down eluate volume) were used for pre-miRNA (~2.5 pmole) cleavage for 15 min at 37°C. Below, the distribution of isomiRs generated by Dicer or Dicer in complex with TRBP in the 5' and 3' arms of the precursors. Data are mean ± SD for two independent experiments. Other designations are as described in the legend for Figure 1.