FIGURE 6.

Fe65 negatively regulates Notch1 signaling via an E3 ligase Itch. A, HEK293 cells were transfected with expression vectors for 4XCSL-Luc and β-galactosidase, along with ΔEN1 and Fe65 as indicated. After 42 h of transfection, the cells were treated with 10 μm of MG132 for 6 h, after which the cells were lysed, and the luciferase activity was determined. B, HEK293 cells were transfected for 48 h with the vector for siControl or siFe65. After 42 h of transfection, the cells were treated with 10 μm of MG132 for 6 h and the cell lysates were immunoblotted (IB) with anti-Notch1 or anti-Fe65 antibody. Antibody to β-actin was used as a loading control. C, HEK293 cells were transfected with expression vectors for 4XCSL-Luc and β-galactosidase, along with ΔEN1, Fe65 and Itch siRNA as indicated. After 48 h of transfection, the cells were lysed, and the luciferase activity was determined. HEK293 cells were transfected for 48 h with the indicated combinations of expression vectors encoding for Myc-Itch and Itch siRNA. The cell lysates were immunoblotted with anti-Myc or anti-β-actin antibody. Antibody to β-actin was used as a loading control. A and C, the data were normalized with β-galactosidase. The results were expressed as the mean ± S.E. of three independent experiments. R.L.U., relative luciferase units. The cell lysates were immunoblotted with anti-Myc antibody. The data we assessed for significant differences via Student's t test. *, p < 0.01 (analysis of variance).