Twenty CNC explants from stage 18 embryos were incubated in Ca2+- and Mg2+-free MBS for 1 h at 12°C, and the dissociated cells were labeled with 500 μl of a 2 mg/ml sulfo-NHS-LC-biotin solution (Pierce, 21335) for 20 min at 15°C. After three washes with 100 mM of a glycine solution, the cells were lysed in MBS, 1% Triton X-100, 5 mM EDTA and proteinase inhibitors. Following centrifugation at 12,000 g for 30 min at 4°C, immunoprecipitation was performed overnight with 10 μg of anti-Intα5 antibody (P8D4, a kind gift from Dominique Alfandari, University of Massachusetts, MA) loaded on Protein A/G agarose beads (Invitrogen, 20423). The beads were washed three times in Tris-buffered saline (TBS) that contained 1% Triton X-100 and once in TBS, treated with lyases and ultimately eluted in 2× non-reducing Laemmli Buffer. Proteins were separated by 8% SDS-PAGE, transferred onto nitrocellulose and detected with horseradish peroxide (HRP)-conjugated streptavidin (Invitrogen, 1:75,000).
