Fig. 4.

A κB sequence of the ICOSL promoter preferentially binds noncanonical NF-κB members and mediates ICOSL promoter activation. (A) The sequence of an ICOSL κB was aligned with the consensus κB sequence (Upper). EMSA was performed using the ICOSL κB probe and nuclear extracts isolated from nontreated or BAFF- and anti-CD40-stimulated (24 h) M12 cells or from freshly purified WT and NIK KO spleen B cells. (B) A supershift assay was performed using nuclear extracts of BAFF-stimulated M12 B cells and the ICOSL κB probe, in either the absence (none) or the presence of the indicated antibodies or an Ig control. The supershifted bands are indicated by arrows. (C) HEK293 cells were transfected with the indicated NF-κB members, either alone or in combination. Nuclear extracts were subjected to EMSA using the ICOSL κB and a general κB probe. Immunoblot analysis was performed to monitor the expression of the different NF-κB proteins. (D) M12 cells were infected with pGreenFire lentiviral vectors carrying a luciferease gene driven by either WT ICOSL promoter (ICOSL-luc) or mutant ICOSL promoter with mutated κB site (ICOSLΔκB-luc). The cells were either not treated (NT) or stimulated for 14 h with LPS, anti-CD40, or BAFF. Luciferase activity is presented as fold induction compared with the NT pGF cells.