Identification of Notch3 interacting proteins in protein array.

<p>(<b>A</b>) Ingenuity pathway analysis shows that Notch3 interaction proteins are enriched in a network involved in gene expression, cell cycle, and development. Colored symbols: Notch3-interacting proteins identified in our screen. UBC: ubiquitin C. (<b>B</b>) Western blot analysis of GST pull-down was performed with GST-peptide control or recombinant WWP2-GST in the presence of increasing concentrations of rhN3-ICD protein using anti-GST antibody or anti V5 antibody. Lower panel shows western blot analysis of flow through after GST pull-down. Reduced N3-ICD is seen only after pulldown with WWP2-GST but not with control GST-peptide. (<b>C</b>) In vitro ubiquitination experiment was performed using recombinant human Notch3 as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5B), and FLAG-tagged WWP2. Ubiquitinated species of rhN3-ICD were detected by immunoprecipitation with V5 antibody followed by western blot with anti-ubiquitin antibody (upper panel). Western blot analysis with anti-FLAG antibody or anti-V5 antibody was performed to detect total input amounts of WWP2 and N3-ICD (lower panels). Ubiquitinated rhN3-ICD was only detected when WWP2 was present.