Fig. 3. PMA-inducible GPIX reporter activity is dependent on an intact Ets motif. A, 5' nested deletions were constructed to identify the motifs responsible for PMA-inducible GPIX expression in Dami cells. B, site-directed mutagenesis was used to introduce mutations into the GATA or Ets motifs of the GPIX-luciferase reporters. Cells were transfected with 5 µg of the indicated GPIX-luciferase reporter constructs and 1 µg of pRL-TK internal control plasmid. After transfection, each sample was split into two populations and growth-arrested overnight. One population was treated with PMA (100 nmol/liter), and the other was treated with vehicle alone (DMSO). Cell extracts were harvested 2 days later, and luciferase activity was analyzed. GPIX-dependent reporter activity was normalized to Renilla luciferase activity under the control of the thymidine kinase promoter to account for variations in transfection efficiency. , -PMA; , +PMA. Each experiment was performed in duplicate at least three times. Results represent normalized mean ± SD of a representative experiment.