Figure 2.

Reelin cleaves L1. (a) Freshly homogenized reeler hippocampus was treated with supernatants from HEK cells expressing (HEK^rln) or lacking (HEK^Ø) Reelin. (b) HEK^rln and HEK^Ø cells were transfected with wild-type L1 (L1) or mock DNA (−). (c) HEK^rln cells were transfected with wild-type (L1^wt) or mutated (L1^mut) L1. (d) HEK cells were co-transfected with wild-type L1 and wild-type Reelin (rln^wt) or mutated Reelin (rln^mut). (a–d) Representative immunoblots of cell lysates out of six independent experiments using an antibody against the intracellular L1 domain (L1-ICD) and the GAPDH antibody are shown. The cropped blots display all L1 forms or the GAPDH band. Mean values  + SEM from 6 independent experiments are shown for the levels of full-length L1 (L1-FL) and differences between groups are shown (*p < 0.05, **p < 0.0001; One-way ANOVA with Tukey’s Multiple Comparison Test). RI: relative intensity in arbitrary units (AU). (e) Purified brain L1 was incubated with heparin-purified wild-type (RLN^wt) or mutated (RLN^mut) Reelin protein in the absence or presence of the serine protease inhibitor PMSF (+PMSF) and then subjected to immunoblot analysis with L1 antibody recognising the intracellular domain (L1-ICD). (f) Schematic representation of L1 structure and cleavage by Reelin. Shown are the extracellular Ig and FN III domains, the transmembrane and intracellular domain (ICD).