Supplementary Figure 4: Hes1 mutants lose Cxcl1-suppressive capacity.
From: The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation
(a) Schematic drawings of murine Hes1 domains and mutants. 3 amino acids (E43, K44 and R47) in the basic region were mutated to alanine to obtain a dominant-negative form of Hes1 (dnHes1) that cannot bind to DNA but can still dimerize with endogenous Hes1 to form a non-DNA-binding heterodimeric complex. Hes1 (∆HLH) and Hes1 (∆WPRW) mutants were generated by deletion of HLH domain (amino acids 48-92) and the last C-terminal 6 amino acids of Hes1 respectively. (b) Immunoblotting with an anti-FLAG antibody to confirm expression of wild type Hes1 (wtHes1) and Hes1 mutants in HEK293T cells. Cells were transiently transfected with recombinant pMx vectors expressing GFP, wtHes1 or Hes1 mutants. 24 hours post transfection, cells were harvested for immunoblotting analysis. p38 served as a loading control. (c) qPCR analysis of Cxcl1 mRNA expression in BMDMs transduced with retroviral particles expressing GFP, wtHes1 or Hes1 mutants and subsequently stimulated with LPS (10 ng/ml) for 1 h. Data are shown as mean ± s.d. of technical triplicate determinants and are representative of two independent experiments (c).
