After washes, 9EG7 antibody (2 μg/ml in Dulbecco’s modified Eagle’s medium [DMEM]) was applied, followed by incubation for 15 min at 37°C. After washes, the cells were lysed in SDS sample buffer. Bound 9EG7 antibodies were detected using biotin-conjugated donkey antirat IgG (1:2000, Jackson ImmunoResearch) followed by horseradish peroxidase-conjugated streptavidin (1:2000, Perkin Elmer). Detailed procedures are described in the Supplemental Experimental Procedures. Cell adhesion

assay was performed as described previously (Bourgin et al., 2007), with some modification. E16 embryonic cortices were dissociated, stimulated with Reelin-conditioned medium for 15 min at 37°C, and plated onto the coated wells (7 × 104 cells per well) filled with DMEM for 5 min at 37°C. Then, after three washes with warm DMEM, the attached cells were counted (nine microscopic selleck compound fields [20×] were counted in each well). Detailed procedures are described in the Supplemental Experimental Procedures. The fluorescence intensity of GFP and Dylight-549 was detected using FV1000. Detailed procedures are described in the Supplemental Experimental Procedures. For direct comparisons, selleck chemical the data were analyzed by Mann Whitney

U test (n < 10). For multiple comparisons, ANOVA was performed, followed by Tukey's posthoc test. All bar graphs were plotted as mean ± SEM. This project was supported by the Strategic Research Program for Brain Sciences (“Understanding of molecular and environmental bases for brain health”), Grant-in-Aid for Thiamine-diphosphate kinase Scientific Research, Global COE Program of the Ministry of Education, Culture, Sports, and Science and Technology of Japan, and Keio Gijuku Academic Development Funds. J.H. is supported by grants from the NIH, AHAF, the Consortium for Frontotemporal

Dementia Research, and SFB780. We thank Drs. T. Curran (reelin); J. Cooper (dab1); F. Miller (Tα1 vector); D. Turner (mU6-provector); M. Matsuda (c3g); H. Kitayama (rap1a); N. Minato (spa1); L. Huganir (n-cadherin); T. Tsuji (integrin α3); M. Ginsberg (talin1); J. Takagi (2A-reelin); C. Cepko (pCALNL vector); T. Miyata (Tα1-Cre vector); and J. Miyazaki (pCAGGS vector) for providing the plasmids, and all of the members of the Nakajima laboratory for discussion. K.S. is a research fellow of the Japan Society for the Promotion of Science. K.S. designed and performed all of the experiments, analyzed the data, and wrote the manuscript. T. Kawauchi designed the initial experiments, analyzed the data, and wrote the manuscript. K.K. supported the preparation of Reelin, performed some in utero electroporation, and analyzed the data. T.H. constructed a part of the Dab1 expression vectors and analyzed the data. J.H. and M.H. provided the mutant mice, and J.H. edited the paper. T. Kinashi designed the integrin experiments and analyzed the data. K.N. supervised the whole project, analyzed the data, and wrote the manuscript.