A1 is a direct WT1 target. (A) CV1 cells were transiently transfected with either a control plasmid or a plasmid directing the expression of WT1 (–Ex5/–KTS) or WT1 (+Ex5/+KTS) along with a reporter plasmid containing the firefly luciferase gene controlled by the murine A1 promoter. Luciferase assays were normalized by cotransfection with a Renilla luciferase gene. Error bars represent the standard error of triplicate assays. Inset shows a Western blot of lysate from transiently transfected cells. Lane 1 shows control plasmid; Lane 2, WT1 (+Ex5/+KTS); and Lane 3, WT1 (–Ex5/–KTS). The top arrow indicates a nonspecific band that confirms equal protein loads in each lane, and the bottom arrow indicates the position of WT1. MW is the molecular-weight markers. (B) Serial deletion mutants of the A1 promoter were generated as described, and these were cotransfected into CV1 cells along with either the control plasmid or the WT1 (–Ex5/–KTS) expression vector. Luciferase assays were performed as described, and were normalized by cotransfection with Renilla luciferase. Error bars represent the standard error of triplicate assays. □ indicates control; ▪, WT1 (–Ex5/–KTS). (C) The sequence of the 71-bp promoter fragment, with the putative WT1 binding site in bold type. The 19-bp deletion is contained within the brackets. The indicated plasmids (pGL3Basic, which lacks a promoter, the 71-bp promoter fragment, and the deletion mutant of the 71-bp fragment) were transfected into CV1 cells along with either a control plasmid or the WT1 (–Ex5/–KTS) expression vector and luciferase assays were performed. Assays were normalized by cotransfection with Renilla luciferase, and error bars represent the standard error of triplicate assays. □ indicates control; ▪, WT1 (–Ex5/–KTS).