Activity of the PU.1 distal enhancer C/EBP-binding sites in myeloid cells. (A) Diagram of reporter constructs containing point mutations in the C1, C2, C5, or promoter C/EBP-binding sites. DE indicates 237-bp PU.1 distal enhancer; Pr, PU.1 485-bp PU.1 promoter; and LUC, luciferase cDNA. (B) 32DPKCδ cells were transfected with 15 μg of the indicated reporters and 0.5 μg CMV-βGal. Two days later, cell extracts were analyzed for luciferase and β-galactosidase activities. The ratio of these activities for DE was set to 100% in each experiment. Results shown are mean and SE from 6 determinations. (C) The indicated constructs were assayed similarly, with DE linked to wild-type promoter (Pr) set to 100% activity in each experiment. Results are mean and SE from 6 determinations. P values versus DE-Luc (or versus Pr-Luc for mPr) are from the Student t test.