Transcriptional activity of the CD80 or CD86 promoter. (A) Effects of PU.1 on each promoter. (B) Reporter assay with deletion constructs to identify PU.1-responsive elements. CV-1 cells were transfected with 2 μg of reporter plasmid, 2 μg of expression plasmid, and 2 ng of pRL-CMV (Promega) as an internal control of transfection efficiency. After 24 hours of culture, cells were harvested, luciferase activity was measured, and relative luciferase activity was calculated as described previously.²⁴  m, co-expression of mock (pCR-3.1); P, co-expression of PU.1 (pCR-PU.1). (C) Transcriptional activity of the CD80 or CD86 promoter in hematopoietic cells. J774 cells were transfected with 2 μg of reporter plasmid and 125 ng of pRL-null. (D) Effects of IRF4/8 on transactivation activity of PU.1. CV-1 cells were transfected with 2 μg of reporter plasmid, a total of 2μg of expression plasmid (adjusted with mock vector as in our previous study²⁵ ), and 125 ng of pRL-null. CD80-1 (−51/+65), CD86-1 (−98/+46), CD86-2 (−96/+47), and IL12 p40 (−315/+14) were used. m, pCR-3.1; P, pCR-PU.1; 4, pCR-IRF4; 8, pCR-IRF8. Relative luciferase activity is represented as the ratio of activity to that of pGL4-Basic (C) with pCR3.1-mock (A, B, D). Data represent the mean + SD of triplicate samples. A representative result of 3 independent experiments is shown.