Figure 1

Fra-1 and c-Jun are members of DCA induced AP-1 Complex. SKGT4 cells were stimulated with 300 μM DCA for 6 hr (B) or the indicated times (A, C, D). Nuclear extracts (4 μg) were prepared and analyzed for AP-1 binding activity by EMSA (A). For supershift assays the nuclear extracts were incubated for 30 min with 450 ng of antibodies against the various Jun and Fos proteins or with unlabelled probe prior to incubation with the radiolabelled probe. Gastric adenocarcinoma cells (AGS) treated with 50 ng/ml PMA were used as a positive control (B). Total cell lysates were prepared and standardized to 350 μg and DNA affinity purification performed. Affinity purified proteins were resolved by SDS-PAGE and immunoblotted with anti-Fra-1, anti-JunB or anti-c-Jun antibodies, respectively (C). Unbound proteins were immunoblotted with anti-α-tubulin as loading control. Whole cell lysates were standardized to 50 μg and examined by Western blotting as in C (D). Results are representative of at least three independent experiments.