Fig 2: Ago2 binding to all three GW proteins TNRC6A-C is affected by Ago2 S387 mutations (related to Fig 3)HEK293 cells transfected with Flag-tagged TNRC6A, B or C plus GFP, GFP-WT-Ago2, GFP-S387A-Ago2 or GFP-S387D-Ago2 were lysed, and GFP-Ago2 complexes were precipitated using GFP-trap beads. Bound proteins were detected by Western blotting using Flag or GFP antibodies as shown. Graphs show quantification of GFP-Ago2 interactions with TNRC6 isoforms, normalised to GFP-WT-Ago2; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA, Bonferroni post hoc test. Error bars are SEM.Source data are available online for this figure.

Fig 3: Transient increase in GW182-Ago2 interaction and S387 phosphorylation in response to NMDAR stimulation Transient increase in Ago2-GW182 interaction. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, and lysates were prepared 0, 3, 6, 10, 20 min after NMDA washout and immunoprecipitated with Ago2 antibodies or control IgG. Proteins were detected by Western blotting. The inputs are shown in (B). Graph shows quantification of Ago2-GW182 interaction, normalised to vehicle control; n = 4. **P < 0.01, ***P < 0.001; one-way ANOVA, Bonferroni post hoc test. Mean ± SEM.Transient increase in S387 phosphorylation and Akt activation. The same lysates from (A) (1% of input) were analysed by Western blotting using antibodies against pS387 Ago2, Ago2, pS473 Akt, Akt, GW182 and GAPDH as a loading control. Graphs show quantification of pS387 Ago2 levels normalised to total Ago2 (top) and pS473 Akt normalised to total Akt (bottom); n = 4. *P < 0.05; two-way ANOVA, Bonferroni post hoc test. Mean ± SEM. Source data are available online for this figure.

Fig 4: Endogenous Ago1-GW182 and Ago1-DDX6 interactions are unaffected by NMDAR stimulation (related to Fig 1)Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min; lysates were prepared 10 min after NMDA washout and immunoprecipitated with Ago1 antibodies. Proteins were detected by Western blotting. Graph shows quantification of Ago1-GW182 interaction, normalised to vehicle control; n = 5; t-test.Source data are available online for this figure.

Fig 5: Ago2 association with GW182 in neuronal dendrites increases in response to NMDAR stimulation Endogenous Ago2-GW182 and Ago2-DDX6 interactions increase in response to NMDAR stimulation. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, and lysates were prepared 10 min after NMDA washout and immunoprecipitated with Ago2 antibodies. Proteins were detected by Western blotting. Graph shows quantification of Ago2-GW182 interaction, normalised to vehicle control; n = 5. *P < 0.05; ***P < 0.001; t-test; mean ± SEM.Analysis of endogenous Ago2-GW182 co-localisation in cortical neuronal cultures. Cortical neuronal cultures were exposed to NMDA or vehicle for 3 min, fixed 10 min after NMDA washout, permeabilised and co-stained with Ago2 and GW182 antibodies. Representative whole-cell images are shown. Scale bar = 50 µm.Endogenous GW182-Ago2 co-localisation increases in response to NMDAR stimulation in neuronal dendrites. Images show dendrites taken from boxed region in (B), above. Graph shows Pearson's co-localisation coefficients; n = 4 independent experiments (18–24 cells per condition). *P < 0.05, t-test. Scale bar = 10 µm. Mean ± SEM.Line-scan analyses of Ago2 and GW182 fluorescence intensities in control and NMDA-stimulated dendrites shown in (C).NMDAR stimulation has no effect on endogenous Ago2-GW182 co-localisation in neuronal cell bodies. Images show cell bodies taken from boxed region in (B). Graph shows Pearson's co-localisation coefficients; n = 4 independent experiments (18–20 cells per condition), t-test. Scale bar = 10 µm. Mean ± SEM. Source data are available online for this figure.