The following plasmids were used for transcriptional transactivation assays: pRc/RSV (Invitrogen, Carlsbad, California, United States), pRc/RSV-Er81, pRc/RSV-EWS-Pea3, pTP-5xETS, and pTP-5xETSmut. pRc/RSV-Er81 and pRc/RSV-EWS-Pea3 were obtained by insertion of the cDNAs for Er81 or EWS-Pea3 (gift from J. A. Hassell) into pRc/RSV. pTP-5xETS was constructed by inserting a cassette of five repetitive copies of high-affinity Pea3 binding sites (5′- GCCGGAAGC-3′) [18,19] into a modified version of pTK-Luc. pTP-5xETSmut was generated as pTP-5xETS but using a mutated complement of the Pea3 binding sites (5′- GCCTATGGC-3′). A control plasmid to normalize for transfection efficiency (placZ) and pTK-Luc were a gift from D. Kressler. COS-7 cells were co-transfected with 1–1.2 μg of total DNA including one of the effector plasmids pRc/RSV-empty, pRc/RSV-Er81, or pRc/RSV-EWS-Pea3; one of the reporter plasmids pTP-5xETS or pTP-5xETSmut; and placZ. Cells were harvested after 25 h and processed for assays to determine luciferase and LacZ activity as described previously [44]. Luciferase values normalized to LacZ activity are referred to as luciferase units.