Fig. 10.

ZEB1 attenuates TGF-β-mediated activation of transcription from Zp. AGS cells in 24-well plates were cotransfected with 80 ng of pZp-Luc-WT or a mutant variant of it containing the base pair substitution mutations in the ZV and ZV′ elements of Zp indicated in Fig. 4B (i), 350 ng of pcDNA3.1 or 100 ng each of pCS2-FLAG-Smad2, pCS2-FLAG-Smad3, and pCMV6-XL5 Smad4 and 50 ng of pcDNA3-ALK5 T204D (ii), and the indicated amounts of a ZEB1 expression plasmid, pCi-ZEB1, plus pcDNA3.1 for a total of 12 ng DNA (iii). Luciferase activities were measured after incubation for 2 days. Data were internally normalized to the amount of protein in each lysate and externally normalized to basal activity observed for each reporter in the absence of the Smads, Alk5 CA, and ZEB1. Bars indicate means ± SD of data obtained from assays performed in quadruplicate. Results presented here are typical of data obtained on four separate occasions.