Nascent protein synthesis in E7 DRG neurons. The methionine analog AHA in methionine-free DMEM was added to dissociated cells treated with 40 ng/ml NGF (a), 0.5 μg/ml Sema3A (b), or control medium (cont) (c) for 60 min. Cells in d received no AHA. Cells were fixed and incubated with fluorescent alkyne to label the AHA incorporated into nascent proteins. e, Mean staining intensity of NGF- and Sema3A-treated distal axon segments relative to unstimulated neurons. Mean ± SEM. *p < 0.05, **p < 0.001. f–i, The protein synthesis inhibitors CHI and anisomycin (aniso) inhibit protein synthesis. Dissociated DRG cells cultured overnight in medium with 10 ng/ml NGF were incubated 60 min in methionine-free DMEM with AHA (f) or with addition of 20 μm CHI (g) or 20 μm anisomycin (h). Cells in i received no AHA. Fluorescent alkyne labeling of the CHI- and anisomycin-treated neurons and axons (f, g) was as weak as labeling of cells that were not incubated with AHA (h). Scale bars: a (for a–d), 10 μm; f (for f–i), 15 μm. j, Mean staining intensity of distal axons. Mean ± SEM. *p < 0.00001.