Figure 4. Hes1 binding to Ptf1-p48 correlates with functional inhibition of the PTF1 transcriptional complex.

(A) Schematic representation of in vitro transcribed and translated ³⁵S-labelled Hes1 proteins used in the GST pull-down assays with full-length GST–Ptf1-p48. (B) Retention of full-length and truncated versions of ³⁵S-labelled Hes1 by GST–Ptf1-p48 and GST alone, as determined by Phosphorimager analysis following resolution on SDS/15% PAGE gel. The input lanes were loaded with one tenth of the total ³⁵S-labelled Hes1 used in each pull-down assay. Full-length Hes1, Hes1 (bHLH+O+C) and Hes1 (bHLH+O+C+W) were retained by GST–Ptf1-p48, whereas versions of Hes1 lacking either the C-terminal [Hes1 (N+bHLH)] or bHLH [Hes1 (O+C+W)] domains were not retained. (C) Corresponding effects of full-length and truncated versions of Hes1 on the activity of the PTF1 transcriptional complex were assessed by activation of PTF-responsive luciferase reporter (PTF1-luc). Maximal inhibition of PTF1 activity was achieved by full-length Hes1, Hes1 (bHLH+O+C), and Hes1 (bHLH+O+C+W), whereas versions of Hes1 unable to bind Ptf1-p48 had a lesser effect.