FIGURE 4.

EGF induces the complex formation and the binding of c-Jun·ARNT to the COX-2 gene promoter. A–C, cells were starved for 18 h in serum-free culture medium and then treated with EGF for various time periods (as indicated) in the culture medium without serum. Cellular lysates (A) and nuclear extracts (B) of cells were prepared and subjected to Western blot or immunoprecipitated (IP) with antibodies against c-Jun and ARNT bound to protein A-agarose. The proteins were subjected to SDS-PAGE and analyzed by Western blotting with anti-c-Jun and anti-ARNT antibodies. C, confluent cells were starved for 18 h in serum-free culture medium and then treated with EGF for various time periods (as indicated) in the culture medium without serum. Nuclear extracts were prepared, and DNA affinity precipitation assay was performed, as described under “Experimental Procedures.” Binding of c-Jun and ARNT proteins to CRE probes was analyzed by Western blot. The streptavidin-agarose beads were used to serve as a nonspecific binding control. D, cross-linked chromatin derived from EGF-treated cells was immunoprecipitated with c-Jun and ARNT antibodies and analyzed by PCR with specific primers for the region from –186 to +49 bp of the COX-2 promoter. Input, nonimmunoprecipitated cross-linked chromatin.