Figure 1. Nuclear Notch ICD is reduced in cortical neurons in Reeler.

(A-H) Nuclei (red) and Notch1 (green in A-D’, antigen is Notch1 intracellular domain) and Notch1 ICD cleaved form (green in E-H) immunostaining in the cortex of wild-type and mutants as indicated. Higher magnifications are shown in the insets. Dashed lines in (E-G) indicate the pial surface and the border between CP and MZ in wild-type or superplate (SPP) in the mutants. (G) and (H) show upper CP and lower CP/IZ, respectively (the asterisk indicate the same cell). Note that Notch1 ICD expression at the lower CP (inverted layer II/III) is significantly decreased in Vldlr/ApoER2 mutant (H) which is similar to Reeler (not shown). Green staining at the pial surface in (G) is nonspecific. Bars = 25 μm. (I) Immunoblots of full-length Notch1 (p300), Notch1 ICD and β-actin from the same cortical lysates of 2 rl/+ and 2 rl/rl. Notch1 ICD bands include p110 non-phosphorylated and p120 phosphorylated forms. Right panel: Relative values of the band intensity of indicated proteins from rl/rl against those from rl/+, which were set as 100. Band intensities for each mutant were normalized to β-actin. The data represent the mean ± SD of 5 brains from independent experiments. *p<0.01 by paired t-test. (J) Immunoprecipitation (IP) of brain lysates with a non-specific goat IgG as negative control (left) or Dab1-specific antibody (E-19; right) followed by Notch1 and Dab1 immunoblotting (IB).