Cultured neurons were transfected with WT VCP or the R95G mutant at (a–e) 12 DIV and (f,g) 2 DIV, and protein synthesis was analyzed using AHA labeling at 2, 4 or 6 days later, as indicated. AHA labeling was detected with alkyne-modified Alexa Fluor-488. (a,d,f) Representative images of the AHA labeling. The insets indicate the transfected cells, which are either outlined or indicated by arrows in the original images. (b) Relative AHA intensities in the dendrites. (c,e,g) The relative AHA signals in the somata. In (a–c,f,g), AHA was labeled for 1 h; in (d,e), AHA was added to the culture for 4 or 6 h, as indicated. In (f), AHA omission or cycloheximide addition (Chx; 10 μM) was used to confirm the specificity of the AHA labeling. Scale bar: 25 μm. The sample sizes (n) of the analyzed neurons are indicated. The data from three independent experiments are presented as the mean+s.e.m. (error bars). NS, non-significant; **P<0.01; ***P<0.001. Unpaired t-test (b,c,e); one-way ANOVA (g).