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Fig. 1. Netrin and DCC induce UNC5 tyrosine phosphorylation. (A) Netrin-induced tyrosine phosphorylation of UNC5 in spinal cord neurons. Spinal cords were dissected from E15 mouse embryos. Neuron cultures were starved for at least 4 hours in serum-free DMEM medium. Netrin (200 ng/ml) was added to the indicated well for 5, 10 and 20 minutes. Lane 2 was pretreated with PP2. Neurons were lysed in PLC buffer and immunoprecipitated with anti-UNC5 antibody. Phosphotyrosine antibody was used to detect phosphorylated UNC5 protein. IB, immunoblot; IP, immunoprecipitation; pY, anti-phosphotyrosine blot. (B) DCC is required for netrin to induce UNC5 tyrosine phosphorylation. UNC5 was transfected with or without DCC in HEK293 cells as indicated. Transfected cells were serum-starved for 8 hours before stimulation. Netrin was added to the indicated lane for 5, 10 and 20 minutes and cells were lysed in PLC buffer. Tyrosine phosphorylation of immunoprecipitated UNC5 was detected by anti-phosphotyrosine immunoblot. (C) The intracellular domain of UNC5 is required for morphological changes in COS-7 cells. COS-7 cells were transfected with DCC wild-type, UNC5 wild-type and their intracellular domain deletion mutants as indicated. After a 24 hour transfection, transfected cells were treated with AP-netrin for 30 minutes. Alkaline phosphatase stained for AP-netrin, which bound to UNC5- and/or DCC-expressing cells (dark color). Bar, 100 µm.
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