The tissue was centrifuged again, HBSS was removed, and the tissue immediately frozen at

−80 °C and stored until used for Western blot analysis. Reelin-treated and control spinal cord tissue was dissected and lysed in ice-cold RIPA lysis buffer containing 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, pH 7.4, 1 mm EDTA, 1% NP-40, 0.5% Na-deoxycholat, 0.1% SDS, 0.004% NaN3 with protease inhibitor and phosphatase inhibitors. The lysates were centrifugated at 10 000 g twice for 20 min at 4 °C. The resulting crude supernatants were taken, and protein concentration was measured by using the DC Protein Assay (BioRad, Munich, Germany). Equal amounts BIBF 1120 of protein in sample buffer were loaded and separated by SDS polyacrylamide gel electrophoresis. Proteins were transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare, Munich, Germany). The membranes were blocked in Tris-buffered solution (TBS), pH 7.4, with 0.05% Tween20 (TBS-T) and 5% non-fat dry milk. Membranes were washed three times using TBS-T and incubated overnight at 4 °C with primary antibodies diluted in TBS-T containing 5% BSA. Membranes were washed three times for 5 min with TBS-T following incubation with the secondary antibody diluted in TBS-T containing 5% BSA for 1 h at room temperature.

Signals were detected by enhanced chemiluminiscence with SuperSignal West Pico Chemiluminiscent Substrate (Pierce Protein Research Products, Thermo Fisher Scientific, Rockford, IL, USA) on Fuji Super RX film. Photographs were either taken with an Olympus BX 61 or Zeiss LSM 510 NLO confocal microscope. Images were processed using Adobe Photoshop 5.5. As a first step in our study of a BIBW2992 mw potential role of Reelin-induced cofilin phosphorylation for normal arrest of SPNs in the IMLC, we retrogradely traced these cells by labelling them with DiI in embryonic tissue from wild-type animals,

reeler mutants and mutants lacking the Reelin receptor VLDLR. As shown next previously (Yip et al., 2003, 2007a,b, 2009), retrogradely labelled SPNs in wild-type animals were found in ventral and dorsolateral positions at E13.5, reflecting their migratory route from the neuroepithelium near the central canal to ventrolateral and then dorsolateral locations, eventually assembling in the IMLC (Fig. 1A). In reeler mice, DiI-labelled SPNs were similarly observed in ventrolateral positions; however, their assembly in the IMLC was incomplete, as reflected by the weak fluorescence staining of the IMLC (Fig. 1B). Instead, many SPNs could be traced to more medial positions (Fig. 1B, arrow), suggesting an ‘over-migration’ of SPNs towards the central canal. A much less pronounced phenotype was observed in vldlr mutants of this embryonic stage (Fig. 1C). In adult mice, the normal assembly of SPNs in the IMLC and the result of aberrant migration in reeler and Reelin receptor mutants were visualized by retrograde labelling with FG (Fig. 2A–D).