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. 2013 Feb 15;110(10):4117–4122. doi: 10.1073/pnas.1217050110

Fig. 2.

Fig. 2.

SGS3 specifically associates with miR173–RISC when RISC binds to TAS2 target RNA in vitro. (A) Association of NtSGS3a-myc with FLAG-NtAGO1 in the presence of miR173/miR173* and TAS2 RNA. FLAG-NtAGO1 was immunoprecipitated from the BYL-based reaction mixtures containing FLAG-NtAGO1, NtSGS3a-myc, 50 nM miR173/miR173*, and 50 nM target RNAs with anti-FLAG antibody. The immunoprecipitates were analyzed by Western blotting (WB) with anti-myc or anti-FLAG antibodies. The input samples were analyzed in parallel. (B) Association of FLAG-NtAGO1 and NtSGS3a-myc with cleaved fragments of target RNA. FLAG-NtAGO1 and NtSGS3a-myc were immunoprecipitated with anti-FLAG and anti-myc antibodies, respectively, from BYL-based reaction mixtures containing FLAG-NtAGO1, NtSGS3a-myc, 50 nM miR173/miR173*, and 5 nM 32P-labeled target RNAs. RNA was extracted from the input samples and immunoprecipitates and analyzed by denaturing 5% PAGE and autoradiography. As controls, FLAG-LUC, LUC-myc, and TAS2-171 RNA were used in place of FLAG-NtAGO1, NtSGS3a-myc, and TAS2 RNA, respectively. (C) Association of NtSGS3a-myc with slicer-defective FLAG-NtAGO1. Experiments were performed as described for B, except that FLAG-NtAGO1D857A defective in slicer activity was used. (D) Formation of a complex containing RISC, target RNA, and SGS3. Experiments were performed as described for B, except that immunoprecipitation was performed first with anti-FLAG antibody, followed by a second immunoprecipitation with anti-myc antibody. The band marked “NS” shows an in vitro transcription by-product that does not have the miR173 target site. The structures of the target RNAs and sRNAs are shown in Fig. S3.