Figure 2A Tethering assay using the F-Luc-HhR-poly(A) reporter and λN-HA-AGO1 (wild-type or pocket mutants) in S2 cells. A plasmid expressing R-Luc served as a transfection control. (A) F-Luc activity and mRNA levels were normalized to those of the R-Luc transfection control and set to 100% in cells expressing the λN-HA peptide. The panel shows mean values ± standard deviations from three independent experiments. B Northern blot of representative RNA samples corresponding to the experiment shown in (A). C,D Tethering assay using the R-Luc-HhR-poly(A) reporter and λN-HA-AGO2 (wild-type or mutants) in humanHEK293T cells. A plasmid expressing F-Luc served as a transfection control. R-Luc activity and mRNA levels were normalized to those of the F-Luc transfection control and analyzed as described in panels (A,B). E,F Tethering assay using the F-Luc-HhR-A95-C7-HhR reporter and λN-HA-AGO1 (wild-type or mutants) in S2 cells. Samples were analyzed as described in panels (A,B). The panel shows mean values ± standard deviations from three independent experiments. E,F Tethering assay using the F-Luc-HhR-A95-C7-HhR reporter and λN-HA-AGO1 (wild-type or mutants) in S2 cells. Samples were analyzed as described in panels (A,B). The panel shows mean values ± standard deviations from three independent experiments. G Western blot analysis showing the equivalent expression of the λN-HA-AGO1proteins used in the tethering assays shown in panels (A,B,E,F). H,I Tethering assay using the R-Luc-HhR-A95-MALAT1 reporter and λN-HA-AGO2 (wild-type or mutants) in HEK293T cells. H,I Tethering assay using the R-Luc-5BoxB-A95-MALAT1 reporter and λN-HA-AGO2 (wild-type or mutants) in HEK293T cells. J Western blot analysis showing the equivalent expression of the λN-HA-AGO2 proteins used in the tethering assays shown in (C,D,H,I). K,L Complementation assays using F-Luc-par6 reporter in S2 cells depleted of endogenous AGO1. Plasmids encoding HA-AGO1 (wild-type or mutants) or HA-MBP (as negative control) and miR-1 primary transcript (or empty vector) were included in the transfection mixtures as indicated. For each condition, firefly luciferase activities and mRNA levels were normalized to those of the Renilla luciferase transfection control and set to 100% in the absence of miR-1 (100% values are only shown for control cells). (K) Normalized firefly luciferase activities and mRNA levels. (L) Northern blot of representative RNA samples. In all panels, bars represent mean values and error bars represent standard deviations from at least three independent experiments. M A tethering assay using the F-Luc-HhR-poly(A) reporter was performed in S2 cells. The transfection mixtures included plasmids expressing GFP or GFP-DCP1* as indicated. The panel shows a northern blot of representative RNA samples. The positions of the polyadenylated (An) and deadenylated (A0, dashed red line) mRNA reporter are indicated on the left. N A tethering assay using the β-globin-6xMS2 reporter and MS2-tagged proteins was performed in HEK293T cells. The transfection mixtures contained plasmids expressing wild-type DCP2 (-) or the catalytic DCP2* mutant (+). The panel shows a northern blot of representative RNA samples analyzed as described in (panel M).