Fig. 3: USP28 interacts with NICD independently of the NICD PEST domain and FBXW7.

A HEK293 cells were co-transfected with FLAG-USP28 S67D/S714D and different NICD variants with different levels of truncation from the side of the C-terminus (upper panel). Co-immunoprecipitation (lower panel) was performed 24 h after transfection and analyzed by western blot of which a short (upper panel) and long (second panel from top) exposure are shown. Expression of the protein derived from the transfected constructs was detected via western blot shown in the two lower panels. * marks the heavy chain of the anti-FLAG antibody used for immunoprecipitation. B NOTCH1 wt (left panel) or NOTCH1 ko (right panel) HEK293 cells were transfected with the indicated FLAG-tagged FBXW7α constructs with complete or truncated WD40 domain which is important for interaction with NICD. Additionally, untagged USP28 was transfected. Co-immunoprecipitation was performed 24 h after transfection and analyzed via western blot shown in the upper panel (IP). The expression of the protein derived from the transfected constructs is shown in the lower panels (Input). * marks the heavy fragment of the anti-FLAG antibody used for IP. C Immunoprecipitation of endogenous NOTCH1 from HG3 wt or the HG3 CRISPR/Cas9-modified FBXW7 WD40 domain knockout cell lines D8, D40, D13 and D24 [17]. Co-immunoprecipitation of USP28 was analyzed via western blot (upper panel, IP). The expression of FBXW7, USP28 and NOTCH1 in the cell lines is shown in the lower panels (Input). Specificity of the endogenous NOTCH1 precipitation was confirmed by IP reactions with only beads or an IgG2b isotype control antibody. * marks the heavy chain of antibodies used for IP. Western blots are representative for at least three independently performed experiments. IP immunoprecipitation, WB western blot, wt wild type.