A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function

A Representative co-immunoprecipitation experiment in hippocampal lysates from adult rat using a pan-Ago antibody. Upper panel: anti-Ago2 Western blot demonstrates specific pull-down of Ago2 in pan-Ago IP. Lower panel: anti-Nova1 Western blot demonstrates specific co-precipitation of Nova1 (52 kDa) and Ago proteins. *Non-identified cross-reactive proteins.

B RNA immunoprecipitation (RIP) in adult rat hippocampus using an anti-Nova1 antibody. Left: specific IP of Nova1 with the applied antibody was confirmed by Western blot analysis. *Non-identified cross-reactive proteins. Right: qPCR for the indicated transcripts is presented as the ratio of mRNA present in Nova1 or control IgG IPs compared to the input material. Data is the average ± standard deviation from three independent experiments. **P < 0.005 (unpaired Student’s t-test).

C Luciferase assay in cortical neurons (five DIV) transfected with a reporter gene which harbors the Limk1 3′UTR fused to a destabilized firefly ORF (Limk1-lucPEST) and with the indicated shRNAs. Neurons were stimulated with BDNF (100 ng/ml) or mock-treated for 5 h before processing for luminometry. Values were normalized to a co-transfected Renilla reporter and the basal condition without shRNA was set as 1. Data is the average ± standard deviation of four independent experiments. *P < 0.05 (unpaired t-test). ns: not significant.

D–F Luciferase assay in cortical neurons (five DIV) transfected with RL-5boxB (tethering plasmid) together with plasmids expressing the indicated NHA-fusion proteins. Values were normalized to a co-transfected firefly reporter, and a basal condition without additional NHA protein was set as 1. Data is the average ± standard deviation of four independent experiments. One-way ANOVA: P < 0.0001 (D, F), P < 0.005 (E); Tukey’s HSD test: **P < 0.01; ns: not significant.

Figure 5.