Fig. 8. Notch pathway activation inhibits activity of the Ptf1 transcriptional complex. (A) Electrophoretic mobility shift assay measuring endogenous Ptf1 DNA-binding activity in nuclear extracts of rat AR42J cells infected with adenoviral vectors encoding GFP alone or in combination with either Notch1-IC, Hes1, Hey2 or Hey1 as indicated. Upper band (arrow) represents specific binding of oligonucleotide corresponding to the A element of rat elastase promoter, confirmed by supershift in the presence of anti-Ptf1-p48 antiserum (arrowhead). Cold indicates presence of excess unlabelled oligonucleotide. Note inhibition of endogenous Ptf1 DNA-binding activity by Notch1-IC, Hes1 and Hey1. (B) Western blot analysis of COS7 extracts following co-transfection of Ptf1-P48 alone or in combination with E47 and either Notch1-IC, Hes1, Hey2 or Hey1. Note constant level of Ptf1-p48 and E47 protein across conditions, allowing analysis of Ptf1 activity (C) in the absence of associated changes in component protein levels. (C) Corresponding determination of Ptf1 activity as assessed by activation of Ptf1-responsive luciferase reporter (Ptf1-luc). Notch1-IC, Hes1, Hey2 and Hey1 effectively inhibit Ptf1 activity, independent of associated changes in protein levels of Ptf1-p48 or E47. (D) Schematic depiction of Notch1-IC truncation mutants used in E and F, below. NLS indicates nuclear localization signal. ANK indicates ankyrin repeats. (E) Ability of Notch1-IC and Notch1-IC truncation mutants to activate Su(H)-responsive luciferase reporter [Su(H)-luc]. (F) Corresponding ability of Notch1-IC and Notch1-IC truncation mutants to inhibit Ptf1-p48/E47-mediated activation of Ptf1-responsive luciferase reporter (Ptf1-luc). Ability to inhibit Ptf1 resides within N-terminal domains. (-) in C,E,F indicates transfection of reporter construct alone.