Fig. 3

Truncation of RELA reveals TWIST1 binding domain is also required for IL-8 regulatory activity. a Schematic representation of RELA alleles. b CoIP shows that expression of truncation mutants of either TWIST1 or RELA prevents most binding between TWIST1 and RELA. Co-expression of both truncation mutants further reduces binding, validating the truncated domains as required binding sites for their counterpart proteins. RELA ∆307 bands have been shown separate from WT due to difference in electrophoretic mobility on account of reduced size. c Quantitation of duplicate western blots following CoIP of TWIST1 with indicated RELA alleles. ∆307 mutation reduced protein binding by 90% on average. Graphed is the ratio of TWIST1 to RELA, each normalized to their respective inputs. d Dual luciferase assay reveals that while the Δ307 allele of RELA reduces TWIST1-mediated upregulation of IL-8 when compared to WT RELA, the same trend is seen in the absence of TWIST1. This suggests that the C-terminal portion of RELA is required not only for TWIST1 binding, but also for proper transcriptional activity. Graph represents firefly luciferase expression normalized to renilla luciferase for each condition. Error bars represent standard deviations of biological triplicate experiments. WT TWIST1 + WT RELA condition was used as the basis for statistical comparisons. pGL3 lacking the IL-8 promoter was used as a negative control. ***, p < .001; ****, p < .0001