(a-c,h) Rat cultured hippocampal neurons were transfected with plasmids, as indicated, and subjected to analyses of (a) ER morphology, (b,h) protein synthesis based on AHA labeling and (c) neuronal morphology using GFP signals. Transfected neurons are indicated either by arrowheads or yellow outline. ER signals in (a) were processed with the Surpass Mode of the Imaris software (Bitplane). (d) Quantification of the D/S ratio of DsRed-ER. (e,i) Relative intensities of AHA-Alexa fluor 488 in soma. (f) Quantitation of dendritic protrusion densities. (g) Co-immunoprecipiation of VCP and ATL1 from transfected COS-1 cells. Full-size images are available in Supplementary Fig. 9. (j–m) GFP-actin was cotransfected with various plasmids into cultured neurons as indicated to analyze the density of dendritic spines. (j) Atl1 expression partially rescued the effect of Vcp knockdown. (k) The ATL1 R217Q mutant and VCP R95G mutant did not have an additive effect on dendritic spine density. (l) Expression of the RAB10 T23N mutant further reduced the density of dendritic spines in VCP R95G-expressing cells. (m) Extra leucine increased the dendritic spine density of ATL1 R217Q mutant-expressing neurons. Scale bar: (a–c,h) 20 μm. The data from three independent experiments are presented as the mean+s.e.m. (error bars). Cumulative probability distributions of spine density are also shown. The sample sizes (n) are indicated. **P<0.01; ***P<0.001; NS, non-significant. One-way ANOVA (d,e,f); two-way ANOVA (i–m).