Figure 7.

Binding of PP2B, c-Jun, and Sp1 to genes promoter. (A and B) Confluent cells were starved for 18 h in serum-free culture medium and then treated with 15 μM CsA for 30 min, followed by 5 nM PMA treatment for 2 h. Nuclear extracts from CsA- and PMA-treated cells were prepared and DNA affinity precipitation assay was performed as described under Materials and Methods. Binding of Sp1, c-Jun, and PP2B proteins to Sp1 probes were analyzed by Western blot. The streptavidin-agarose beads were used to serve as a nonspecific binding control. (C) Cross-linked chromatin derived from PMA-treated cells was immunoprecipitated with PP2B, c-Jun, and Sp1 antibodies and analyzed by PCR with specific primers for 12(S)-lipoxygenase promoter [12(S)-LOX], cPLA2α promoter, and thrombomodulin promoter (TM), which was used as a negative control. Input, nonimmunoprecipitated cross-linked chromatin. Similar results were obtained in two or three independent experiments.