1.   Nuclear extracts were prepared as previously described.

2.   Nuclear extracts were either treated or mock-treated with phosphatase and then analysed using the blotting assay.

3.   Nuclear extracts were also immunoprecipitated with anti-CREB antibody or pre-immune serum and probed as above.

4.   Nuclear extracts were also immunoprecipitated with anti-CREB antibody and probed by Far Western.

5.   Nuclear extracts were also immunoprecipitated with CREB antibody or proteins purified by sequence-specific DNA-affinity chromatography and probed as above.

6.   The two c-Jun derivatives were then tested for an ability to be phosphorylated by kinases present in a crude HeLa cell nuclear extract.

7.   Taken together, these data indicate that a DNA-dependent kinase present in crude HeLa nuclear extracts specifically phosphorylates the bZIP region of c-Jun.

8.   This protein was then purified and tested for an ability to be phosphorylated in a crude HeLa cell nuclear extract.

9.   The Jun-Core S A protein also failed to display DNA-dependent phosphorylation when assayed in nuclear extracts.