Figure 6. Lipid rafts are not required for the GDNF-induced ubiquitination or degradation of Ret. A, Sympathetic neurons were treated with MCD (10 mm), or medium alone, for 15 min, washed, and then stimulated with GDNF (50 ng/ml) or medium alone for 1 h. The neurons were extracted with 1% Triton X-100, and the insoluble pellets (left) containing lipid rafts were collected via centrifugation from the soluble, nonraft fractions (right). These fractions were separated by SDS-PAGE and immunoblotted with antibodies to Ret51. To confirm the relative purity of the insoluble and soluble fractions, flotillin and transferrin receptor immunoblotting, respectively, was conducted. B, Neurons were treated with MCD as in A, washed, and then stimulated with GDNF (50 ng/ml), or medium alone, for 3 h. Whole-cell extracts were then produced from the neurons, and the extracts were subjected to Ret9 (top) and Ret51 (2nd panel) immunoblotting. Actin immunoblotting confirmed that equal amounts of protein were analyzed. C, Sympathetic neurons were treated with medium alone or with neutral Smase (2 mU/ml) for 90 min. The neurons were then stimulated with GDNF (50 ng/ml) or vehicle alone for 1 h in the continued presence of Smase. These neurons were then subjected to Triton X-100 extraction and immunoblotting as described in A. D, Similar to the treatments described in C, sympathetic neurons were treated with Smase before treatment with GDNF or medium alone for 3 h. Total cellular protein was harvested, and analyzed as was done as in B. E, Cells were treated with MCD, Smase, or vehicle only, as done in the previous panels. After a 15 min treatment with GDNF (50 ng/ml), the neurons were detergent extracted, and ubiquitinated proteins were immunoprecipitated. These immune complexes were analyzed with Ret51 immunoblotting, and actin immunoblotting of the supernatants confirmed that equal amounts of protein were analyzed. The experiments in this figure were performed two to four times with similar results. INSOL, Insoluble pellets containing lipid rafts; SOL, soluble fraction containing nonraft membrane fractions; IP, immunoprecipitate; TrfR, transferrin receptor; Cav, caveolin; W, Western blot; WCL, whole-cell lysate.