Fig. 1. Ligand-independent partitioning of DCC in lipid membrane rafts. (A) HEK293 cells were transfected with a DCC-expressing construct and 24 hours after transfection, cell were treated or not with either MßCD or CO as described in the Materials and Methods. The cell lysates were solubilized in Brij 98 and subjected to sucrose gradient separation. Immunoblots performed on the different sucrose fractions were revealed with HRP conjugated anti-DCC, anti-Fyn or anti-TfR antibodies, or with cholera toxin B-HRP (GM1). (B) Same as A but Triton X-100 was used instead of Brij 98. (C) HEK293 cells were transfected with netrin-1 and/or DCC-expressing constructs and 24 hours after transfection cell lysates were solubilized in Brij 98 and subjected to sucrose gradient separation. Immunoblots performed on pooled heavy fractions (8 and 9) and light fractions (1-4) were revealed with HRP-conjugated anti-netrin-1 and anti-Rab5 antibodies. (D) DCC is in lipid rafts in commissural neurons. Same as in C but 3 x10⁶ commissural neurons dissociated from rat E13 embryos were used instead of HEK293 cells. Raft, raft containing fraction; HF, heavy fraction. Raft inhibitors MßCD (10 mM for 12 minutes) or CO (2 U/ml for 1 hour) were included in the incubation at and just before the PNS preparation.