Site-specific mutation of the Sp1 binding sites between positions -67 to -85 of theJak3promoter or a 2-base change in the -44 to -53 GAS motif markedly reducesJak3pr activity in M1 cells. (A) The -100 Jak3pr construct was used as a template to create -100 Sp1 mut, in which 3 consecutive Sp1 consensus sites are destroyed by making a 2-base change in each site. Additionally, -100 Stat mut was created by making a 2-base change to disrupt the -44 to -53 GAS motif. (B) -100 Sp1 mut and -100 Stat mut were transiently transfected into 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. Values for the -100 wild-type construct were normalized to 100 for M1 cells stimulated with IL-6 and normalized to 50 for unstimulated M1 cells.