Inhibition of PU.1 activity by GATA-1.
(A) Structure of the PU.1 responsive pPU3-TK-LUC luciferase reporter. (B) pPU3-TK-LUC (1 μg) was cotransfected into Q2bn fibroblasts in the presence (solid bars) or absence (hatched bars) of 0.25 μg of CMV–based PU.1 expression vector (pCMV-MTPU.1) or empty pcDNAI expression vector as indicated. Increasing amounts of GATA-1 expression vector (pSPCMV-GATA-1) were titrated into the experiment. Empty pSPCMV expression vector was added to a total of 1 μg. The luciferase activity was measured and is shown normalized to the β-galactosidase activity obtained from the pRSV-βgal internal control plasmid (0.25 μg); the values are expressed relative to that obtained with pPU3-TK-LUC in the presence of empty expression vectors only (lane 1). The data shown are the average of 2 determinations from a representative experiment. (C) Equal amounts of total cell lysate (corresponding to about 1 × 105 cells) from cells transfected as in (A) were electrophoresed on a 12% SDS-PAGE gel and subjected to Western analysis with anti–GATA-1 antiserum (panel a) or 9E10 monoclonal antibody (detecting the MycTag on the PU.1 protein; panel b). Arrows indicate the positions of the full-length GATA-1 and PU.1 proteins. (D) pGATA-1P-LUC (1 μg) was cotransfected with the indicated amounts of pSPCMV-GATA-1 expression vector and pRSV-βgal internal control plasmid (0.25 μg) and fold activation calculated as described in (B).
