Fig. 3. FAK is required for netrin-induced UNC5 tyrosine phosphorylation. (A) Netrin fails to stimulate UNC5 tyrosine phosphorylation in FAK^-/- MEF. UNC5 was transfected with DCC into wild-type and FAK-knockout MEF cells. 36 hours after transfection, cells were serum-starved for about 8 hours followed by netrin stimulation (200 ng/ml) for 20 minutes. UNC5 protein was immunoprecipitated by anti-HA antibody. Immunoprecipitates were subjected to anti-phosphotyrosine immunoblot. (B) Expression of FAK restores the ability of netrin to stimulate UNC5 tyrosine phosphorylation. FAK (10 ng) was co-transfected with UNC5 and DCC into FAK^-/- MEF cells. The experiment was conducted under similar conditions to those described in panel A.