NRTN (30 nmol) and 2 nmol of GFRα2 and RET or mutants thereof were incubated at 4°C for 16 hours. BS3 cross-linker (Thermo Fisher Scientific) was added at 0- and 2500-fold molar excess, and the samples were incubated for 30 min at room temperature. The reaction was stopped by adding tris-HCl (pH 8.0) to a final concentration of 50 mM, followed by incubation at room temperature for 15 min. The samples were analyzed by 4 to 12% SDS-PAGE analysis and Coomassie staining.

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