To identify the major E3 ligase for c-Myc ubiquitylation in HEK293T cells, we overexpressed FLAG-tagged c-Myc in HEK293T cells and treated with MLN4924. and analyzed the interactors by immunoprecipitation and MS. Cell lysates were subjected to immunoprecipitation with antibodies to FLAG, and the immunoprecipitated proteins were analyzed by LC-MS/MS.