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

Figure 4.

Figure 4

Both PACT and TRBP are associated with the slicer activity in vitro. (A) In vitro pre-miRNA processing assay and the detection of mature miRNA bound to the RISC components. Dicer, hAgo2, TRBP, or PACT fused to FLAG was expressed in HEK293T cells and immunoprecipitated in RNAi reaction buffer. The immunoprecipitates were incubated with 5′-end radiolabeled pre-miR-30a. The left panel shows the RNA extracted from each reaction tube. After in vitro processing, the beads were washed with RNAi reaction buffer and treated with phenol to extract the RNA associated with the proteins (right panel). (B) In vitro RNAi assay. Dicer, hAgo2, TRBP, or PACT fused to FLAG was expressed in HEK293T cells and immunoprecipitated in RNAi reaction buffer. The immunoprecipitates were incubated with cold pre-miR-30a or 27mer siRNA. After washing, the immunoprecipitates were incubated with 5′-end radiolabeled target RNA that is complementary to miR-30a-5p.