Fig. 4

Competitive inhibition of TWIST1 WR domain binding. a Schematic representation of GFP alleles used. GFP contains 23 amino acids encoded by the multiple cloning site of the vector at its C-terminus. GFP-WR contains the first two such amino acids (Leu-Glu encoded by XhoI restriction site), followed by the 20 amino acids of the WR domain. Thus, the two alleles have indistinguishable molecular weights. b Left, fluorescent microscopy shows that GFP and GFP-WR are expressed at similar levels and in similar patterns in HEK-293 cells. Scale bar, 100 μm. Right, Western blot confirms equal GFP and GFP-WR expression. c CoIP with RELA pulldown reveals that in the presence of increasing GFP-WR expression, TWIST1-RELA binding is reduced in a dose-dependent manner. d CoIP with GFP pulldown reveals that increasing GFP-WR dose results in more TWIST1 co-precipitated with GFP. Graph represents ratio of TWIST1 to GFP, normalized to their respective inputs. Error bars, standard deviation of duplicate experiments. e Dual luciferase assay demonstrates that as seen in the RELA CoIP, there is a dose dependent drop in IL-8 driven luciferase expression with increasing dose of GFP-WR inhibitor. Graph represents firefly luciferase expression normalized to renilla luciferase for each condition. Error bars represent standard deviation of biological triplicate experiments. GFP without GFP-WR condition was used as the basis for statistical comparisons. pGL3 lacking the IL-8 promoter was used as a negative control. ****, p < .0001