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. 2012 Jan 13;24(1):259–274. doi: 10.1105/tpc.111.092718

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© 2012 American Society of Plant Biologists. All rights reserved.

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Figure 5. — Detection of Inhibition Effects of SD2b and Its Mutations on AGO1-Mediated Cleavage Activity in Vitro.

(A) Effects of SD2b and its mutations (as indicated above the gel) on endogenous sRNA/Flag-AGO1–mediated cleavage activity in vitro. Flag-AGO1 obtained from inflorescences of Flag-AGO1/ago1-36 plants were incubated with buffer (lane 1) or GST-tagged SD2b or mutant proteins (lanes 3 to 12) and 32P-cap–labeled in vitro transcripts of PHV. Similar immunoprecipitated extracts from wild-type plants served as negative controls (lane 2). 32P-cap–labeled PHV RNA and 5′ cleavage products are indicated.

(B) Immunoprecipitates containing Flag-AGO1 protein were detected by immunoblot using a monoclonal antibody to Flag (top panel), and coimmunoprecipitated miR165 was detected by RNA gel blotting.