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. 2019 Apr 17;20(7):e46436. doi: 10.15252/embr.201846436

Figure 2. NICD and FBXW7 interact directly at endogenous levels.

Figure 2

  1. NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 μg of HEK293 cell lysates treated with DMSO or MLN4924 was subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by Western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β‐Actin served as loading control.
  2. Quantification of the density of Western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student's t‐test was used to determine P values, with **P ≤ 0.01.
  3. Roscovitine treatment reduced the NICD‐FBXW7 interaction. 500 μg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine was subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by Western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β‐actin served as loading control.
  4. Quantification of the density of Western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924‐treated samples. All data represent the mean ± SEM from three independent experiments. Student's t‐test analysis was performed, with ***P ≤ 0.001.