Figure 7: MICAL-1 regulates IgCAM cell surface expression to control mossy fibre lamination in vivo.

(a) Immunohistochemistry for PSA–NCAM or CHL1 in coronal sections of P55 mice. Staining was performed in the presence or absence of detergent to visualize total and cell surface expression, respectively. SP, stratum pyramidale. n=6 MICAL-1^+/+, n=5 MICAL-1^−/−. Scale bar, 90 μm. (b) Subcellular fractions of the hippocampus of P10 mice were subjected to western blot analysis using the indicated antibodies. Each experiment contained an internal loading control (actin). Representative examples are shown, full blots in Supplementary Fig. 9. Numbers at the right indicate kDa. H, homogenate; KO, MICAL-1 knockout; P3, membrane; S3, cytosol; WT, wild type. (c) Quantification of band intensity of experiments as shown in (b) was performed by scanning densitometry. Signals were normalized to internal actin control and data were expressed as % of WT in each experiment. Graph shows means±s.e.m. from at least three independent experiments. Neo, neogenin; Nrp2, neuropilin-2. (d) Immunostaining of coronal sections of the hippocampus of P55 mice of the indicated genotypes using anti-NF antibodies (red) together with DAPI staining (blue). Scale bar, 140 μm. (e) Quantification of the genetic interactions between MICAL-1, NCAM and CHL1. n=5–7 mice for each genotype. P<0.001 compared with single heterozygous mice, one-way ANOVA. Data are presented as means±s.e.m.