Fig. 4.

Gel mobility shift assay for the USP16 gene promoter. Gel shift and Super gel shift assays were performed as described in the Materials and methods. a-d Double-stranded consensus NFκB oligonucleotides were end-labeled with IR700 dye as probes. Incubation of labeled probe with nuclear extracts formed a shifted DNA-protein complex band (lane 2). For competition assays, different concentrations of unlabeled competition oligonucleotides, consensus NFκB (lanes 3), mutant NFκB (lanes 4), USP16 NFκB (lanes 5 and lanes 6), USP16 NFκB mutant (lanes 7) were added. Anti-NFκB p65 antibody was used for the super gel shift assay. Addition of the anti-NFκB p65 antibody into the reaction mixture produced a supershifted band, indicating the formation of the nuclear protein-USP16-p65 complex (lane 8). e-g USP16 NFκB2, USP16 NFκB3, and USP16 NFκB4 double-stranded oligonucleotides were end-labeled with IR700 dye as probes, respectively. Incubation of labeled probe with nuclear extracts formed a shifted DNA-protein complex band (lane 2). For competition assays, unlabeled competition oligonucleotides, USP16 NFκB (lanes 3), USP16 NFκB mutant (lanes 4), consensus NFκB (lanes 5), mutant NFκB (lanes 6) were added. Anti-NFκB p65 antibody was used for the super gel shift assay. The anti-NFκB p65 antibody supershifted the nuclear protein-USP16-p65 complex (lane 7). h A double-stranded oligonucleotide contains USP16 NFκB2, USP16 NFκB3, and USP16 NFκB4 cis-elements were end-labeled with IR700 dye as probe for EMSA