Fig. 5

EXT1 regulates NOTCH1 degradation through FBXW7. a Protein complementation assay in HEK cells transfected with NICD-Gluc1 and/or FBXW7-Gluc2, in addition to EXT1-yfp as indicated in the X-axis, Normalized Luciferase Value (NLR) is represented by the Y-axis. b, (c) and (d) HEK293 cells were transfected with NICD-Flag and EXT1-YFP expressing plasmids as indicated. Twenty-four hours post-transfection cells were treated with proteasomal inhibitor MG132 for 6 h and lysates analyzed by western blot using anti-Flag M2 and anti-GFP antibodies. In (d) 24 h before overexpression, cells were transfected with siRNA for FBXW7 or a siRNA control using calcium phosphate. e Relative mRNA expression levels of FBXW7 analyzed by qPCR. Data represent the means ± SEs of three independent experiments, each performed in triplicate