Fig. 4. Src family kinases are important for UNC5 tyrosine phosphorylation in response to netrin. (A) PP2 inhibits netrin-stimulated UNC5 tyrosine phosphorylation. UNC5 and DCC were transfected into HEK293 cells. Before netrin treatment, PP2, a Src family kinase inhibitor, was added to the culture for 30 minutes. The phosphotyrosine immunoblot showed that PP2 inhibited netrin-induced UNC5 tyrosine phosphorylation. (B) Src enhances UNC5 phosphorylation. UNC5 was transfected with wild-type Src kinase (lanes 2 and 3) and Src (K/M), a kinase-dead mutant (lane 4). Lane 3 was treated with PP2. Anti-HA immunoprecipitates were subjected to immunoblotting with phospho-tyrosine antibody.