Figure 4
Rapid NMDAR-dependent increase in interaction of endogenous DDX6 with Ago2 protein and Limk1 mRNA, but not Apt1 mRNA. (A) NMDAR-dependent interaction of DDX6 with Ago2. Neurons were stimulated with NMDA or vehicle for 3 min, followed by 10 min incubation after NMDA washout. Cells were lysed and subjected to immunoprecipitation with anti-DDX6 or with control IgG. RNA and proteins were isolated by trizol extraction, bound mRNAs were quantified by qPCR and bound proteins were detected by Western blotting with anti-DDX6 or anti-Ago2. Left panel shows representative blots, graph shows quantification of Ago2-DDX6 interaction. n = 5 independent experiments, *p < 0.05, t-test. Data are mean ± S.E.M. (B) Quantification of Limk1 and Apt1 mRNA bound to DDX6 or to IgG control from IPs shown in A. Values are IP/input ratios, normalised to vehicle control. *p < 0.05, n = 6 independent experiments, two-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M. (C) Ago2-DDX6 interaction increases rapidly after NMDAR stimulation. Neurons were stimulated with NMDA or vehicle for 3 min, followed by washout and a range of incubation times. The time points stated in the figure are the time after the start of NMDA application. Cells were lysed and treated as in A. Left panel shows representative Western blots, graph shows quantification of Ago2-DDX6 interaction over time. n = 5 independent experiments, *p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M. (D) Limk1, but not Apt1 mRNA shows rapid and transient increase in binding to DDX6 in response to NMDAR stimulation. Graphs show quantification of Limk1 and Apt1 mRNA bound to DDX6 at each time point after NMDAR stimulation. Values are IP/input ratios, normalised to vehicle control. n = 5 independent experiments, *p < 0.05, one-way ANOVA followed by Tukey’s multiple comparison test. Data are mean ± S.E.M.
