Figure EV3. Luciferase assays using 3′UTRs carrying mutations to block miRNA binding (related to Fig 5).

• A–E
  The effects on luciferase reporters incorporating LIMK1, APT1 and LIN41 3′UTRs seen in Fig 5 are fully explained by regulation via miR‐134, miR‐138 and Let‐7, respectively. Cultured cortical neurons transfected with Ago2 molecular replacement constructs as well as Renilla luciferase and Firefly luciferase reporters containing LIMK1 (A, D), APT1 (B, E) or LIN41 (C) 3′UTRs containing mutations in the seed regions for miR‐134, miR‐138 and Let‐7, respectively, were treated with NMDA or vehicle for 3 min. Ten minutes after NMDA washout, lysates were prepared for dual‐luciferase assays. For (D, E), cultures were also treated with kinase inhibitors as shown 20 min before NMDA or vehicle. Error bars are SEM.
• F, G
  Translation of luciferase reporters incorporating PUM2 or CREB1 is regulated by other miRNAs in addition to miR‐134. Experiment was performed as in (A), except Firefly luciferase reporters contained PUM2 (F), CREB1 (G) 3′UTRs carrying mutations in the seed regions for miR‐134. n = 5. ***P < 0.001, two‐way ANOVA, Bonferroni post hoc test. Error bars are SEM.