(a) 3D SIM images of cultured hippocampal neurons. DsRed-ER and Myc-tagged WT Vcp or the R95G mutant were cotransfected into neurons at 12 DIV and analyzed with immunostaining using Myc, DsRed and the endogenous ER marker calreticulin (CRT) antibodies at 18 DIV. (b–i) DsRed-ER was cotransfected with the indicated plasmids into cultured neurons. The Z-series of the confocal images were processed with the Surpass Mode of the Imaris software (Bitplane) as shown in (b,e,h). Arrows identify the examples of the dendritic branch sites with enriched ER. (c,f,i) The ratio of dendritic DsRed to somatic DsRed (D/S) and the total number of dendrites. (d,g) The cumulative probability of dendritic DsRed-ER shows the accumulation of DsRed-ER in the proximal region of the dendrites in P47i- and R95G-expressing cells. Ctrl, non-silencing control. Scale bars: (a) original, 10 μm; enlarged, 2 μm; (b,e,h) original, 20 μm; enlarged, 2 μm. The data are from three independent experiments and are presented as the mean plus s.e.m. (error bars). The sample sizes (n) of the examined neurons are indicated. *P<0.05; **P<0.01; ***P<0.001. Unpaired t-test (c,f); two-way ANOVA (d,g,i).