Specific protein binding of ETS/PU.1 and IRE/ISRE consensus sequences of IL12RB1 promoter. (A-B) RAW264.7 cells were left untreated or were treated with IFN-γ, IL-15, or LPS for 45 minutes, and the nuclear extracts were prepared for EMSA. Two micrograms nuclear extract was incubated with 32 P-labeled oligonucleotides complementary to the indicated consensus elements. DNA-protein complexes were separated on a 6% polyacrylamide gel. C1 and C2 indicate protein complex binding to ETS/PU.1; C3, protein complex binding to IRE/ISRE consensus sequence; n.s, nonspecific complex; comp, EMSA binding reaction containing 20-fold excess of the specific cold competitor. (C) Identification of IRF binding to the IRE/ISRE site of the IL12RB1 promoter. Cold oligonucleotides used in the competition assays are indicated. None comp indicates EMSA binding reaction that contained no competitor. (D) Supershift assay using anti-PU.1 antibody (αPU.1 Ab), anti-IRF3 antibody (αIRF3 Ab), or control isotype antibody (C-Ig Ab). EMSA was carried out as in panel A. For supershift experiments, nuclear extracts were preincubated with 2 μg indicated antibody for 1 hour before 32 P-labeled probe was added. Modified DNA-protein complexes are indicated.
