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. 2006 Jan 19;25(3):522–532. doi: 10.1038/sj.emboj.7600942

Figure 1.

Figure 1

PACT interacts with Dicer, but does not facilitate the cleavage activity. (A) Immunoprecipitation followed by Western blotting. V5-Dicer protein was coexpressed in HEK293T cells with FLAG-PACT, or FLAG-TRBP protein. Immunoprecipitation was carried out using anti-FLAG antibody and the protein was visualized using either anti-V5 or anti-FLAG antibody. (B) In vitro pre-miRNA processing assay. Total HEK293 T extract (total lysate) or immunoprecipitates (FLAG-IP) of FLAG-Dicer, FLAG-TRBP, or FLAG-PACT were incubated with 5′ end radiolabeled pre-let-7a-1. The RNA was extracted by phenol extraction method, followed by separating on 15% urea-PAGE. (C) In vitro processing assay using recombinant human Dicer and PACT. In all, 200 ng of recombinant PACT prepared from E. coli and 0.25 U of recombinant human Dicer (Stratagene) were used in each reaction. Pre-let-7a-1 (left panel) and long dsRNA of 80 bp (right panel) were used as substrates.