Figure 3. CREM α binds on SYK promoter.

A, The SYK gene promoter sequence from initiator element to −782. The transcription start site is marked as red; the CRE site on SYK promoter as blue; one AP1 site marked as green; Ets core binding sites as pink. B, Sonicated chromatin from normal T cells was immunoprecipitated by CREM α antibody or a control IgG. Amplified SYK promoter was detected by PCR. C, An oligonucleotude spanning the region of CRE site on SYK promoter was labeled with [γ³²P] and incubated with 5 µg T cell nuclear extract. Protein-DNA complexes were separated on a 6% DNA retardation gel and visualized by phosphoimage analyzer. Unlabeled cold competitors (20 to 500 molar excess) were used to displace complexes formed by labeled oligonucleotides. A cold mutated oligonucleotide was used as non specific competitor. One representative experiment out of 3 is shown. D, 2 µg of specific antibody against CREM α or a non-specific IgG were incubated with labeled oligonucleotides and EMSA was performed. E, Mutated CRE site-bearing oligonucleotide was labeled with [γ³²P] and EMSA was performed. In a separate reaction a wild-type probe was used. One representative experiment out of 3 is shown.