Ubiquitination site mapping of NICD-interacting proteins in mouse by IP-MS

Primary murine γδ T cells were isolated and polarized toward a pro-inflammatory γδT17 phenotype by stimulation with recombinant cytokines IL-1β (20 ng/mL), IL-23 (10 ng/mL), and IL-7 (10 ng/mL) for 72 hours. The Notch intracellular domain (NICD)-associated protein complex was then immunoprecipitated from these activated γδ T cells using an anti-NICD antibody. The immunoprecipitated material was subjected to ubiquitin remnant motif (K-ε-GG) enrichment and analyzed by high-resolution LC-MS/MS on a Q Exactive HF-X mass spectrometer to identify ubiquitination sites on NICD-interacting proteins in the context of γδT17 cell activation.