Enforced CREB and ATF-2 expression trans-activates ^Gγ-promoter activity. (A) Western blot analysis showed increased phosphorylated and total CREB1 and ATF-2 protein levels after enforced expression for 12 and 24 hours after transfection. (B) The effect of increasing concentrations of pCMV-CREB on ^Gγ-promoter transcription was analyzed. Maximal trans-activation was observed at the 50-μg concentration at 24 hours. The base vector was transfected to control for nonspecific trans-activation. The bracket and star above the graph (comparison between the untransfected K562 cells [0] versus those treated with pCMV-CREB) represent significance at the P < .05 level. Data are shown as the mean ± SEM. (C) The effects of enforced ATF-2 (pDNA3.1ATF-2) expression on ^Gγ-promoter activity were tested. A robust concentration-dependent increase in luciferase activity was observed for both expression vectors. (D) The -1500^GγLuc, -1350^GγLuc, -1180^GγLuc, and -1500^AγLuc reporter plasmids (10 μg) were cotransfected into K562 cells with the constitutively active pCMV-CREB expression vector; luciferase activity was analyzed after 24 hours. A pattern of luciferase activity similar to that obtained with the HDACIs was observed. The star above the graph (comparison between untreated and pCMV-CREB-transfected cells) represents significance at the P < .05 level.