Sp1 and PU.1 act synergistically in cotransactivating transcription of the murine MR promoter. Fragments of the murine MR promoter, in which the Sp1 site or each of the four PU.1 sites (separately or in combination) were mutated, were transfected into Drosophila SL cells (Sp1 and PU.1 negative) by the calcium phosphate precipitation method of Chen and Okayama.37 Ten micrograms of the mutated MR plasmid construct was transfected along with 1 μg of each expression plasmid (PUpECE and pPacSP1) and luciferase activity was measured as described in the Materials and Methods. Wild-type (WT) plasmid alone or together with PUpECE and/or pPacSp1 was transfected as a control. The figure portrays the luciferase reporter gene results as a fold increase over activity obtained from transfection of the wild-type (WT) construct.