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Fig. 4. Netrin-1-induced DCC-mediated ERK-1/2 activation is dependent on DCC association with lipid rafts. (A) Wild-type DCC- or mutant C1121V DCC-expressing HEK293 cells were pre-treated or not for 1 hour with 2 U/ml of CO and then treated for 15 minutes with purified netrin-1. Phosphorylated ERK-1/2 was compared with total ERK-1/2 according to the Face ERK-1/2 kit instructions. An index of ERK-1/2 phosphorylation is presented as the ratio between netrin-1-treated cells and untreated cells. (B) Dissociated E13 commissural neurons were pre-treated or not for 1 hour with 2 U/ml of CO and then treated for 15 minutes with purified netrin-1. To confirm the role of DCC in the ERK-1/2 phosphorylation observed in commissural neurons, a blocking DCC antibody was also added ({alpha}-DCC) as previously described (Forcet et al., 2002). Phosphorylated ERK-1/2 was compared with total ERK-1/2 according to Face ERK-1/2 kit instructions. An index of ERK-1/2 phosphorylation is presented as the ratio between netrin-1 treated cells and untreated cells. (C) Same as A except than ERK-1/2 activity was analysed by an in vitro MBP kinase assay following ERK-1/2 immunoprecipitation.