(a) Schematic of mouse Vcp genomic organization and design of the R95G knockin-targeting construct. Dark box and open box indicates WT and VcpR95G (R95G) mutated exons, respectively. (b) Genomic Southern blotting analysis with the 5′ probe: 7.1 kb for WT and additional 2.7 kb for Vcpwt/R95G (R95G) mice; 3′ probe: 7.1 kb for WT and additional 6.2 kb for R95G mice. (c) Genotyping by PCR with primer set 1: products of 475 bp for WT and additional 711 bp for R95G mice; primer set 2: products of 497 bp for WT and 500 bp for R95G allele. The R95G PCR product was digested with EcoRV to generate 236 and 264 bp fragments. In (b,c), images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 9. (d) Sequencing results of the PCR product amplified by primer set 2 to reveal R95G and EcoRV mutations. (e) TEM analysis of cultured hippocampal neurons from WT and R95G mice at 18 DIV. Neurons were collected from nine mice for each group from three independent experiments. Number of analyzed neurons: WT, 120; R95G, 132. Error bars present the mean+s.e.m.. N, nucleus. (f,g) The length and ribosomal density of rER were quantified. Scale bar: 500 nm. ***P<0.001; unpaired t-test.