FIGURE 3.

CREM binds to the −21 CRE site of the CD86 promoter. a, The wild-type CD86 promoter luciferase construct has been cotransfected into U937 cells together with an antisense CREMα (AS), a CREMα expression plasmid (S), a CREB expression plasmid or a control plasmid (EV) and cells were stimulated with LPS for 24 h. Luciferase activity was measured and relative luciferase activity was normalized to luciferase activity of the control plasmid. b, The wild-type (WT) or the mutated (M) CD86 luciferase construct was transfected into U937 cells together with either an antisense CREM (AS) plasmid or a control (EV) plasmid and stimulated with LPS. Luciferase activity was measured 24 h after stimulation with LPS (n = 4) and values were normalized to transfection with the control plasmid. c, CD86 promoter luciferase construct has been transfected into U937 cells together with an antisense CREMα (AS) construct or an empty vector (EV) and reporter ChIP has been performed with Abs against CREM and CREB. DNA was amplified with primers specific for the CD86 luciferase construct and run on a 1.5% agarose gel (n = 3). d, CD86 luciferase construct was mutated at the −21 CRE site (M) and transfected into U937 cells. As control, the wild-type luciferase construct (WT) was transfected and an reporter ChIP was performed with Abs against CREM and CREB. DNA was amplified with primers specific for the CD86 luciferase construct and run on a 1.5% agarose gel (n = 3).