Fig 2: Reelin pathway expression in the hippocampus of rats during the development of SE. (A) Representative immunohistochemical staining of reelin protein in hippocampal tissues during the three phases of SE development. Left: representative immunohistochemical staining of reelin protein (Scale bar=50 µm); right: High-magnification bright-field images and arrows indicating the immunopositive cells (Scale bar=10 µm). (B) Bar graph results of reelin protein in hippocampal tissues during the three phases of SE development. (C) Representative immunohistochemical staining of p-Dab1 protein in the hippocampus. Left: representative immunohistochemical staining of p-Dab1 protein (Scale bar=50 µm); right: high-magnification bright-field images and the arrows indicate the immunopositive cells (Scale bar=10 µm). (D) Bar graph results of p-Dab1 protein in the hippocampus. (E) Western blot analysis for p-Dab1. β-actin was used as an endogenous control. (F) Quantification of western blot results of p-Dab1, presented as the p-Dab1/Dab1 ratio. Data are presented as the mean ± standard deviation. ***P<0.0001, **P<0.001 by two-way analysis of variance with Tukey's post hoc test (n=3 rats/per group). Ep, epilepsy; Con, control; Dab1, disabled 1; p-Dab, phosphorylated Dab1.

Fig 3: Altered pattern of expression of the human Disabled homologs, Dab1 and Dab2, in patients with chronic myeloid leukemia. (A) Downregulation of Dab1 RNA expression in patients with chronic myeloid leukemia (CML) compared to the expression in healthy donors (CTRL). In particular we found a 1 log reduction of Dab1 expression in both peripheral blood (PB) (P<0.01) and bone marrow (BM) (P<0.01) (median values 2−ΔΔct: 0.02 versus 0.3 in PB and 0.008 versus 0.04 in BM). (B) Expression pattern of Dab1 in CML patients during molecular remission (MR) compared to that in treatment-resistant patients. (C) Immunofluorescence staining of Dab1 protein (red) in PB samples of healthy donors, CML patients at diagnosis and CML patients during MR. Nuclei are stained in blue. (D) Quantification of Dab1 protein expression in the immunofluorescence assay. (E) A 3H-thymidine proliferation assay showing a 20% reduction of cell proliferation in K562 cells transfected with Dab1 plasmid compared to control. (F) Western blot of protein extracts from K562 cells transfected with an empty vector (lane 1) and transfected with a Dab1 expression vector (lane 2), showing detectable expression of Dab1 only in K562 cells transfected with the Dab1 vector. Independent loads of equal amounts of protein extract were probed with antibodies raised against BCR, Dab1 and GAPDH as a loading control. (G) Downregulation of Dab2 RNA expression in CML patients compared to the expression in healthy donors. In particular Dab2 expression was found to be statistically decreased (P<0.0001 and P<0.0001 in PB and BM, respectively) with median values of 0.12 versus 2.8 and 0.12 versus 0.7 in PB and BM, respectively. (H) Pattern of expression of Dab2 in CML patients during MR compared to that in treatment-resistant patients. (I) Immunofluorescence staining of Dab2 protein (red) in PB samples of healthy donors, CML patients at diagnosis and CML patients during MR. Nuclei are stained in blue. (J) Quantification of Dab2 protein expression in an immunofluorescence assay. The statistical comparisons were conducted using a Student t test (*P<0.05, **P<0.01, ****P<0.0001). Bars indicate the standard error.

Fig 4: The RELN-H3448R variant enhances Dab1 signaling and the affinity of CTR-RELN to heparin.a, Representative western blotting of pDab1 levels (top) and total protein staining (bottom) levels in primary mouse cortical neurons treated with full-length WT RELN or RELN-H3448R, mouse ortholog of RELN-H3447R (mock, P < 0.0029 and WT RELN, P = 0.0246). Data are presented as the mean ± s.e.m. and were analyzed using a Kruskal–Wallis test with Dunn post hoc analysis for multiple comparisons of n = 4 independent biological experiments. b, Spectroscopic analysis of heparin chromatography fractions of WT CTR-RELN (blue plot) and the CTR-RELN-H3447R mutant (green plot) eluted at increasing gradients of NaCl (0.05 M NaCl step gradient). Data are expressed as the percentage of input over 0.4–5 M NaCl gradient fractions. Data show that 0.55 M NaCl can displace WT CTR-RELN binding from a heparin column. The affinity for heparin of CTR-RELN increases in the presence of the H3447R mutation, as suggested by the shift of the peak with maximum height of the eluted fraction from 0.55 to 0.7 M NaCl. n = 3 independent chromatography experiments. The error bars represent the s.e.m. c, Representative sensorgrams of the binding analysis between chip sensors coated with heparin and 0–25 nM increasing concentrations of CTR-RELN variants. Data are expressed as response units per second. The equilibrium disassociation constant (KD) for each SPR analysis are shown inside the graph and support the difference in affinity binding between heparin and the CTR-RELN variants: H3447R (right plot, KD = 3.75 × 10−9 M-1 s-1) > H3347 (left plot, KD = 6.53 × 10−9 M-1 s-1). The sensorgrams of CTR-RELN with the H3447K and H3447D control variants are reported in Supplementary Fig. 6 for comparison. d, Isothermal calorimetry measurements of short-variant WT CTR-RELN (left) and CTR-RELN-H3447R (right) titrated with 5 μM heparin. Affinity calculations are reported above each plot. e, Binding analysis via BLI between Fc-fusion WT CTR-RELN and H3447R and a heparin-coated biosensor. Association (ka) and dissociation constants (kd) were used to calculate the KD that is displayed in the plot. f, Docking of WT CTR-RELN (purple) with a representative heparin molecule (cyan). Amino acids in CTR-RELN that have polar contacts with heparin are highlighted in magenta.Source data