(a) Increased leucine concentrations in the medium induce the phosphorylation of S6 ribosomal proteins in the cultured neurons. The basal leucine concentration in Neurobasal medium is 0.8 mM. Full-size blots are shown in Supplementary Fig. 9. (b–g) Cultured neurons were transfected with the indicated plasmids at 12 DIV, treated with different concentrations of leucine at 15 DIV and analyzed for (b,c) AHA incorporation, (d,e) ER distribution and (f,g) dendritic spine density at 18 DIV. Full-size cell images of (f) are shown in Supplementary Fig. 7a. In (b), the transfected neurons in the images are either outlined or indicated by arrows. (g) Rapamycin (Rapa; 10 nM) and MNK1 inhibitor CGP57380 (CGP; 10 μM) were added 6 h before harvesting to reduce the beneficial effect of leucine on dendritic spine density. Full-size cell images of (g) are shown in Supplementary Fig. 7b. Scale bars, (b,d) 20 μm; (d), enlarged and (f) 2 μm. The data from three independent experiments are presented as the mean+s.e.m. (error bars). The sample sizes (n) of the examined neurons (c,e) and dendrites (f,g) are indicated. *P<0.05; **P<0.01; ***P<0.001; NS, non-significant. One-way ANOVA (a); two-way ANOVA (c,e,f,g).