Linker-scanning mutagenesis reveals that sequences between positions -43 to -87 are required for activity of theJak3promoter in M1 cells. (A) Shown is a representation of the linker scanning mutants of the Jak3 promoter that was used for luciferase assays. All linker scanning mutants shown above use the Jak3pr -529 construct as a template. The linker scanning mutants span the region between +2 and -150. (B) Linker scanning mutants of the Jak3 promoter and the strongly activating -529 5′ deletion construct were transiently transfected into either unstimulated M1 cells or M1 cells stimulated with IL-6. Following a 20-hour incubation period, cells were lysed and analyzed for luciferase activity. Relative expression refers to the fold increase in luciferase activity over vector alone. Values expressed are the average of 3 independent experiments plus or minus one standard deviation. For M1 cells stimulated with IL-6, the value for the -529 construct was close to 100-fold over empty vector, so this value was normalized to 100 and all other values were expressed as a percentage of the -529 value. For unstimulated M1 cells, the value for the -529 construct was close to 50-fold over empty vector, so this value was normalized to 50 and again all other values were expressed as a percentage of the -529 value.