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. Author manuscript; available in PMC: 2015 Jan 16.
Published in final edited form as: Cell Rep. 2014 Jan 2;6(1):211–221. doi: 10.1016/j.celrep.2013.12.013

Figure 6. Duplex small RNA loading is deficient and RISC loading factors are absent in nuclear extracts.

Figure 6

(A) Cleavage of a 5′-radiolabeled luciferase RNA substrate by Ago2 isolated from cytoplasmic or nuclear fractions treated as shown above the gel. T1, RNase T1 cleavage; OH, alkaline hydrolysis; (+) CTRL, synthetic cleavage product. (B) Co-precipitation of radiolabeled duplex siLuc or radiolabeled single-strand siLuc guide RNA incubated with Ago2 after immunoprecipitation from nuclear or cytoplasmic extracts. (C-D) In vitro assay for Ago2 duplex siRNA loading in extracts. Radiolabeled siRNA is added to extracts from human cell lines, Ago2 immunoprecipitated with anti-Ago2 or anti-FLAG antibody, and co-purified RNA resolved on a denaturing polyacrylamide gel. FHA-A2-c1 and FHA-A2-c3 are two different T47D clonal cell lines stably expressing FLAG-HA-tagged Ago2. (E) Radiolabeled miR-19a or siLuc were used in the same assay shown in panels C and D. Mismatch positions relative to the 5′ end of the guide strand are indicated above the gel. (F) Radiolabeled siLuc was used in the same assay shown in panels C and D but co-purified RNA was resolved on a non-denaturing polyacrylamide gel. Immunoprecipitation wash conditions and shown below the gel and the co-purified duplex or single-strand RNAs indicated to the right. (G) Western blot of RISC loading and maturation factors and subcellular markers from cytoplasmic and nuclear fractions.