Two separate regions in MRE contribute its response to MEKK-1 stimulation. A series of trinucleotide substitution were introduced into -75 to -52 region of both copies of MRE in (MRE)₂/Luc, as shown in the upper panel. These mutant constructs were transfected into VSMCs with pCDNA3.1 blank vector or MEKK-1^*. A dominant-negative AP-1, A-Fos, was also cotransfected with MEKK-1^* to examine the contribution of AP-1. One day after the start of transfection, cells were harvested and luciferase activities were assayed. Luciferase activities were normalized to Renilla luciferase activities of cotransfected pRL-TK. Triplicate transfections were performed in each experiment, and the bar graph shows results from 3 or more separate experiments.