Fig. 1. Slit proteolysis during embryogenesis. (A,B) Slit proteolysis during embryogenesis. (A) Developmental western blot of whole embryo lysate probed with Slit antibody that recognizes both the full-length and carboxyl-terminus fragment, approximately 190 and 50 kDa, respectively. [Note: a previously unreported band of approximately 120 kDa was detected in tissues from early stages. A similar band was also observed in zygotic slit²/slit² null tissues (data not shown). This 120 kDa band is thought to represent either an unknown protein that the Slit antibody recognizes or maternally supplied Slit protein of a previously unreported, truncated form. In the latter case, considering the distance between the Robo-binding domain and the antibody epitope, the protein is unlikely to activate Robo.] (B) Relative concentration of product of Slit proteolysis, i.e. [50 kDa band]/([190 kDa band]+[50 kDa band]). (C,D) Standard immunocytochemistry of wild-type (+/+) embryonic CNS with Slit (C) and Pak (D) antibodies at hour 14. As in other experiments in this study, the embryos were fillet-dissected and fixed. However, from there on they were subjected to 1 mM Triton X-100 throughout the reminder of the process. Embryos were counter-stained with HRP antibody (green). With detergent treatment, Slit can be detected abundantly in its source, the glia, with additional low-level signals outside the source (C, purple). Anti-HRP recognizes extracellular domains of neuronal cell surface proteins and labels axons in both longitudinal connectives (lc) and commissures (ac and pc; C-D, green). However, the cytoplasmic molecule Pak is detected within the entire CNS, including the longitudinal connectives (lc), commissures (ac and pc) and neuronal cell bodies (cb) (D, blue on right). (E,F) Detergent-free immunocytochemistry of wild-type (+/+) embryonic CNS with Slit (E) and Pak (F) antibodies at hour 14. Without detergent treatment, Slit is detected mainly along the longitudinal connectives (lc) and commissures (ac and pc) (E, purple on right; also see Fig. 5). This is a very different pattern from the Slit staining using standard immunocytochemistry (compare with C, purple). By contrast, Pak antibody detects very little Pak protein in either axons or neuronal cell bodies (F, blue compare with C, blue). These observations support the idea that the detergent-free method used in this study detects the pool of Slit protein that exists in extracellular space and excludes the pool that is intracellular. Scale bar: 5 µm.