Figure 6

miR inhibitor interaction with RISC. (a) Immunoprecipitation (IP) of miR inhibitor using AGO2 antibody. PCR primers were used to detect PMIS-miR-17 after pull down, and U6 was used as loading control. Immunoglobulin G antisera was used as a control and PMIS-miR-200c transfected as a PCR control. (b) IP of miR inhibitor using a Dicer antibody. Controls were as in panel a. (c) Schematic of the Dicer and Ago2 pull-down assay. (d) Pull down of AGO2 and Dicer with biotinylated PMIS-miR-17. Biotinylated PMIS-miR-17 RNA or PMIS-miR-17 RNA without the double-stranded regions were incubated with 293 cell extracts, washed, denatured and analyzed by polyacrylamide gel electrophoresis (PAGE). Western blot was performed using Dicer and AGO antibodies. Proteins were detected using ECL reagents (GE Healthcare). (e) Western blot of Dicer after knockdown of Dicer with siRNA. Cell lysates (15 μg) were resolved by PAGE after transfection with siGFP, siDicer 1-1 and siDicer 1-2 (two different siRNA constructs) or water as a control. Western blot was performed using Dicer antibody and β-tubulin antibody as a loading control. (f) Knockdown of Dicer impaired miR inhibitor function. Rluc vector and in vitro transcribed siRNA targeting Rluc was co-transfected with miR inhibitor to siRNA-Rluc and in vitro transcribed siRNA targeting GFP or two different sites of the Dicer transcript. Rluc and Fluc activity was measured 48 h after transfection.