Fig. 6. UNC5 associates with Src. (A) Co-immunoprecipitation of Src and UNC5 in transfected HEK293 cells. UNC5 was transfected with wild-type Src or the kinase-dead Src mutant into HEK293 cells. 48 hours after transfection, transfected cells were lysed and immunoprecipitated by anti-Src antibody. Immunoprecipitates were subjected to anti-HA immunoblot. (B) The SH2 domain in Src is required for the interaction with UNC5. UNC5 was transfected with wild-type Src, SH3 or SH2 Src mutants into HEK293 cells. Coimmunoprecipitation was conducted as described for panel A. Anti-HA immunoblot showed that the Src SH2 mutant significantly decreased the interaction with UNC5. (C) Co-immunoprecipitation of endogenous UNC5 by anti-Src antibody. Brain tissues from E15 mouse embryos were dissected and lysed in NP40 buffer and were used for immunoprecipitation by anti-Src antibody. Immunoprecipitates were subjected to anti-UNC5 immunoblot. Lysate was used as a positive control. (D) Co-immunoprecipitation of endogenous Src by anti-UNC5 antibody. Experiments were similar to panel C. (E) Mapping the region in UNC5 responsible for the interaction with Src. UNC5 recombinant proteins purified from bacteria expressing deletion mutants of UNC5 were used in an in vitro pull-down experiment to test their interaction with Src. Src was immunoprecipitated from transfected HEK293 cells. Src binding on beads was equally divided into each reaction and incubated with different MBP-UNC5 mutants. The UNC5 protein associated with Src was determined by HA immunoblot. UNC5 protein input was shown by Coomassie staining (middle panel).