Fig. 9.

Reduced neuronal ribosome content and inhibition of protein synthesis after knockdown of EMG1 in hippocampal neurons. A, COS7 cells were cotransfected with expression vectors for rat wild type (wt) EMG1-EGFP, β-galactosidase (β-gal) and shRNAs against EMG1 or a control shRNA (shLuc, 1 + 1 + 2 μg of plasmid DNA/60 mm plate, respectively). A mutant variant of rat EMG1-EGFP (mut) that was modified to become resistant to shEMG1–1 was also tested. Western blot for GFP revealed efficient knockdown of rat EMG1wt but not rat EMG1mut 48 h post transfection; the membrane was also probed for β-gal to ensure similar transfection efficiency. B–E, DIV6 rat hippocampal neurons were transfected and stained as described for experiments in Fig. 7B–7E except use of different shRNAs as indicated. B–C, Knockdown of EMG1 reduced mean ribosomal content in the perikarya. D–E, Knockdown of EMG1 reduced general protein synthesis. F–I, Transfections and stainings were as in Fig. 8 except use of different constructs as indicated; pcDNA3.1 was used as an empty vector control (EV) for EMG1-EGFPmut. Note that in F–G, the EGFP expression vector was used instead of RFP (Fig 8C–8D) and ribosomes were stained with NeuroTrace 530/615 Red Nissl Stain to avoid GFP interference. Anti-ribosomal (G) or anti-translational (I) effects of shEMG1–1 are blocked or attenuated by EMG1-EGFPmut. Data represent means ±S.E. of at least 58 cells from three independent experiments (C), 48 cell from two independent experiments (E), 52 cells from two independent experiments (G), or 91 cells from three independent experiments (I); ***, p < 0.001; NS, p > 0.05.