Selective activation of the Elk1

trans-reporter activity by Gab2. (A) K562 cells (2 × 10⁶) were transiently transfected with a total of 4.3 μg DNA consisting of 1 μg pFR-luc, 0.2 μg pCMV-βgal, 0.1 μg pFA2-Elk1 (or pFA2-c-Jun, pFA-CHOP as indicated), and 3 μg pcDNA3.1⁻ or pGab2WT⁺. (B) Cells (2 × 10⁶) were transiently transfected with a total of 4.3 μg DNA consisting of 1 μg pFR-luc, 0.2 μg pCMV-βgal, 0.1 μg pFA2-Elk1, and increasing concentrations of pGab2WT as indicated (total DNA was equalized to 3 μg with pcDNA3.1), or 3 μg Gab2Tyr604Phe. (C) Cells (2 × 10⁶) were transiently transfected with a total of 4.3 μg DNA consisting of 1 μg pFR-luc, 0.2 μg pCMV-βgal, 0.1 μg pFA2-Elk1, and 3 μg pcDNA3.1, 3 μg pGab2WT (Gab2), or 0.5 μg pGab2WT and 2.5 μg pRasN17 (N17). At 18 hours prior to the luciferase assay, the transfected cells were incubated with the indicated concentrations of PD98059 or mock treated with dimethyl sulfoxide (0). At 48 hours after transfection, luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are means and SDs of at least 3 independent experiments performed in duplicate.