Fig. 4.

EGF-induced reporter gene expression is decreased in a dose-dependent manner by cotransfection with Cbl or ICP0. (A) Experimental design: HEK293 cells grown in 12-well plates were transfected with SRE-luciferase reporter gene construct, CMV-LacZ, expression vector pRK-EGFR, and variable amounts of plasmids expressing Cbl or ICP0. The empty expression vector MTS1 was added to the transfection mixture as needed to maintain a constant amount of transfected DNA. After 12 h, the cells were replenished with serum-free medium for 24 h and then incubated in medium containing 100 ng/ml EGF for 4, 8, or 12 h. The cells were then harvested, lysed, and the luciferase and β-galactosidase activities were assayed as described in Materials and Methods. Luciferase activity was normalized against β-galactosidase levels for each transfection, and EGF-induced fold increase in luciferase activity was quantified for every pair in the triplicate and was expressed as the average induction (fold increase) ± SD. (B) Cbl and ICP0 down-regulate EGF-induced reporter gene. EGF-induced luciferase activity in cells transfected with Cbl alone or with ICP0 is shown. (C) ICP0 down-regulates EGF-induced reporter gene. EGF-induced luciferase activity in cells transfected with increasing amounts of ICP0 alone or in combination with Cbl is shown.