Ubiquitinated Ret resides outside of lipid rafts in GDNF-stimulated sympathetic neurons. A, Sympathetic neurons were stimulated with GDNF (50 ng/ml) for various lengths of time, and the neurons were then extracted with Triton X-100. The extracts were centrifuged, and the insoluble pellets containing lipid rafts (left 4 lanes) and the soluble fraction containing nonraft membrane fractions (right 4 lanes) were then separated by SDS-PAGE and electroblotted. The blots were probed with antibodies to Ret9 (top 2 panels), Ret51 (next 2 panels), and PY1062Ret (5th panel). Shorter exposures of the Ret9 and Ret51 immunoblots are shown (labeled as “Light,” panels 2 and 4) to better observe the loss of Ret protein in these fractions. To confirm the relative purity of the lipid-raft fractionation, blots were probed with anti-transferrin receptor (6th panel) and anti-caveolin-1 (bottom panel), which are markers of nonraft fractions and lipid-raft fractions, respectively. B, Sympathetic neurons were treated with GDNF (50 ng/ml) for 15 min and then Triton X-100 was extracted as described in A. The fractions were then treated with glucoside to solubilize the rafts and subjected to ubiquitin immunoprecipitation. The supernatants of this immunoprecipitation were used for a second Ret51 immunoprecipitation, and all of the immunoprecipitates were subjected to Ret51 immunoblotting (top). The activation state of the complexes was determined with phosphotyrosine immunoblotting (second), and the purity of the raft and nonraft fractions was confirmed by probing the supernatants with antibodies to transferrin receptor and caveolin-1 (bottom 2 panels). These immunoblots also confirmed that equal amounts of protein were analyzed. The experiments in this figure were conducted two or three times with similar results. INSOL, Insoluble pellets containing lipid rafts; SOL, soluble fraction containing nonraft membrane fractions; IP, immunoprecipitate; TrfR, transferrin receptor; Cav, caveolin; Ub, ubiquitin; P-Tyr, phosphotyrosine; W, Western blot.
