Effect of patient-specific mutations in FBXW7 on NICD binding. (A-C) Coimmunoprecipitation (CoIP) of NOTCH1-IC (NICD) with wild-type or mutant FBXW7 constructs. HEK293 cells were cotransfected with NICD (which corresponds to the human NOTCH1 intracellular domain, aa 1761-2555) and the indicated Flag-tagged FBXW7α constructs. Expression of NICD (middle panel) and Flag-FBXW7 proteins (bottom panel) was verified by western blotting. CoIPs were performed 24 hours after transfection. ∗The heavy chain of anti-Flag antibody used for IP. Localization of the corresponding mutations is shown schematically in the topmost panel. (B) Lane 8: lysate from untransfected cells, no-CoIP. Control CoIPs for testing unspecific binding to the Flag column are shown in supplemental Figure 2. hs-NICD, Homo sapiens-NICD; IP, immunoprecipitation; WB, western blot.