Figure 1
N-t cleavage of Reelin markedly decreased in the embryonic brain of the PA-DV KI mice. (A) Schematic drawing of the full-length Reelin protein (FL), its cleavage sites (N-t, C-t, and WC), and its cleaved products (NR6, NR2). The white box and black curve indicate N- and C-terminal regions, respectively. The gray ovals indicate Reelin repeats. Binding regions of known Reelin receptors are indicated with lines. The cleavage sites are indicated by dotted lines with arrowheads. The amino acid sequences around the N-t cleavage site of wild-type (WT) and PA-DV KI mice (PA-DV) are shown below the arrow indicating the N-t cleavage site. The changed residues are underlined. WC, cleavage within the C-terminal region. (B) ADAMTS-3 did not cleave the PA-DV sequence. An artificial substrate NR3-MycHis12,60 (lanes 1 and 3) or its PA-DV mutant (lanes 2 and 4) were incubated with the culture supernatant from HEK293T cells transfected with control vector (Mock, lanes 1 and 2) or the ADAMTS-3 expression vector (ADTS-3, lanes 3 and 4) for 16 h at 37 °C. The reaction mixtures were separated by SDS-PAGE and analyzed by western blotting with anti-Reelin G10. The positions of the NR3-MycHis (substrate) and its cleaved product are indicated by arrowheads. The positions of the molecular mass markers (kDa) are shown on the right side of the panels. (C) Western blotting analysis of the indicated regions of the brain of WT (+/+) or PA-DV homozygous (KI/KI) mice at E18.5 using anti-Reelin G10 (top), anti-Dab1 (middle), and anti-β-actin (bottom) antibodies. The position of each protein is indicated by arrows. Each lane represents a sample from an independent mouse of the indicated genotype. The positions of the molecular mass markers (kDa) are shown on the right side of the panels. p-Akt, phosphorylated Akt; t-Akt, total Akt. (D) Quantification of Reelin FL, NR6, and NR2. White and dark-gray bars indicate the amount of each protein in the +/+ and KI/KI mice, respectively. (E) Quantification of Dab1. The white and light-gray bars indicate the amount of Dab1 protein in the +/+ and KI/KI mice, respectively. CC, cerebral cortex; Hip., hippocampus; Cb, cerebellum. (F) Western blotting analysis of Tau phosphorylation. The lysates of the cerebral cortex from the indicated mice were separated by SDS-PAGE and analyzed by western blotting with anti-phosphorylated Tau (AT8, top panel) or anti-Tau (Tau5, bottom panel) antibodies. The positions of the molecular mass markers (kDa) are shown on the left side of the panels. p-Tau: phosphorylated Tau, t-Tau: total Tau. The data are shown as the mean ± SEM and were analyzed by two-tailed Student’s t-test. n = 8. *p < 0.05, ***p < 0.001, and ****p < 0.0001. NS, not significant (p ≥ 0.05).
