Fig. 2From: Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-ProEstablishment of an in vitro RISC assay. a Sequence pairing of MYB33 RNA and miR159 isoforms. The mismatched nucleotides are highlighted in red. b The endogenous AGO1-IP products from Col-0 plant and ago1-27 mutant were incubated with MYB33-230 RNA substrate to perform an in vitro RISC assay. The RNA substrate and cleaved RNA fragments are indicated with arrows. c The AGO1-IP amounts from Col-0 plant and ago1-27 mutant were evaluated by western blotting with α-AGO1 IgG for normalization of the RISC cleavage efficiency. In: input; IP: immunoprecipitation; FT: flow through. d Normalized RISC cleavage efficiency for AGO1-IP from Col-0 plant and ago1-27 mutant. e Sequencing pairing between miR159a and the MYB33mSeed or MYB33mCenter. The mismatched nucleotides are highlighted in red. f In vitro RISC assay for MYB33-230, MYB33mSeed, and MYB33mCenter RNA substrates. The RNA substrate and cleaved RNA fragments are indicated with arrows. (g) In vitro RISC assay for various miRNA-target substrates. h Normalized RISC cleavage efficiency for CSD-230, NAC1-230, and ARF16-230 RNA substrates (i). Normalized RISC cleavage efficiency for ARF10-230 and ARF10-191 RNA substrates (ii). The MYB33-230 RNA substrate served as a positive controlBack to article page