Figure 7: Roquin binds to Ago2.

(a) Immunoprecipitation of HEK293T cells transiently co-transfected with N-terminal V5-tagged Roquin and C-terminal FLAG-tagged Ago2; using anti-V5 and anti-FLAG antibodies. Half of the immunoprecipitate was treated with RNaseA. Roquin and Ago2 proteins were detected by western blot analysis using anti-V5 and anti-FLAG antibodies. Total lysate is shown in lane 1 as input. Mouse anti-CD71 antibody was used as a control. Anti-PABP antibody was used to confirm the RNase activity. The data are representative of three independent experiments. See also Supplementary Fig. 5. (b) Quantification of proximity ligation assays showing average number of PLA spots per average number of cells (PLA/nuclei) for each field of view (represented by the individual dots in each column). At least five fields of view containing 50–100 cells or nuclei were imaged per experiment. Data are representative of four independent assays. U-test: **P<0.002. (c) Confocal images of HEK293T cells showing molecular proximity of endogenous Roquin and Ago2 (left panel), Roquin and RCK (middle panel) and Roquin and GFP (right panel). Nuclei were stained with DAPI. Scale bar, 25 μm. See also Supplementary Fig. 5. (d) Schematic of Roquin binding to miR-146a, Ago2 and Icos 3′-UTR containing CDE and miR-146a binding sites TS1 and TS2. See also Supplementary Fig. 6. (e) Relative luciferase levels in mouse primary T cells retrovirally transduced with either intact full-length mouse Icos 3′-UTR, or this 3′-UTR mRNA carrying mutations in both miR-146a target sites, in the CDE or in both miR-146a target sites plus CDE. Each dot represents a technical replicate. U-test: ***P<0.001; **P<0.01. This is a representative of three independent assays.