Fig. 1. N1-IC inhibits E47 and E12 transcriptional activity by accelerating their degradation. (A) Dose responses. NIH3T3 cells were transiently transfected with 5 µg, 1 µg each of E-box luciferase and β-galactosidase reporter plasmids along with 1 µg of E47 or E12 plus increasing amounts of N1-IC expressing constructs. Luciferase activities were assayed 36 h later and normalized to those of β-galactosidase. Transcriptional activity of E47 in each sample is presented relative to that in the absence of N1-IC with standard deviations from at least three independent experiments. For protein analysis, indicated amounts of E47 or E12 and N1-IC were cotransfected with a GFP expression vector. Whole lysates were probed with anti-E47, GFP and N1-IC antibodies. (B) A proteasome inhibitor blocks the degradation of E47 protein induced by N1-IC. Immunoblot assays were carried out with NIH3T3 cells transfected with 1 µg of E47 expressing plasmid with or without 4 µg of the N1-IC construct and treated with the indicated amounts of MG-132 proteasome inhibitor, leupeptin or dimethylsulfoxide vehicle for 3 h prior to harvest. C indicates loading control. (C) Activation of endogenous Notch receptors enhances degradation of E47. The NIH3T3 fibroblasts expressing Notch ligand, Jagged-1 (J) or not (C), were infected with a retroviral construct producing both E47 and GFP. Cells were harvested 3 days after infection. Whole-cell lysates were analyzed using immunoblotting (IB) with antibodies against E47 and GFP. Nuclear extracts were used for detecting N1-IC. Another aliquot of the infected cells was used to isolate RNA for RT–PCR assays of HES1 and GAPDH mRNA.