Addition of a pool of all the selleck chemicals Gefitinib antibodies completely abrogated the formation of the DCA induced AP 1 complex. These data suggest that Fra 1 and c Jun, but not c Fos, are members of the DCA induced AP 1 complex. To further characterize the composition of the DCA induced AP 1 complex, total cell lysates were prepared from SKGT4 cells treated with 300 M DCA for 1 6 hr and used for DNA affinity precipitation assays with the AP 1 consensus sequence. Only active forms of the Jun and Fos pro teins are able to bind to this oligonucleotide and can be therefore affinity purified and detected by Western blot analysis with specific antibodies. For these assays, we con centrated on c Jun and Fra 1 as suggested by EMSA and also included JunB and JunD, as they have been shown to respectively counteract or enhance c Jun activity.

These experiments show that DCA stimulates a time dependent increase in Fra 1, JunB and c Jun DNA binding activity. No activation of JunD was observed at any stimulation time. DCA induces strong Fra 1 binding activity after 1 hr, which is sustained for at least 6 hr of stimulation. Fra 1 is detected as two bands with distinct electrophoretic mobility a slower migrating, more prominent band and a fainter faster migrating band. After prolonged stimulation, the slower species is stabilized while the faster migrating species is no longer detected. These two elec trophoretic mobility forms of Fra 1, which most likely correspond to different phosphorylation states, have been previously reported in other cell types.

Similarly, DCA induced JunB activity is clear at 1 hr and remains ele vated for up to 6 hr of stimulation. On the other hand, DCA induces a weak and more transient acti vation of c Jun, which is maximal at 4 hr and is consist ently weak and even negligible at 6 hr. These data indicate that DCA induces AP 1 complexes com posed of Fra 1, JunB and c Jun at early stages of stimula tion, but only of Fra 1 and JunB at 6 hrs. Fos and Jun proteins can form heterodimers while the only the mem bers of the Jun family can homodimerise. Therefore, the possible types of early induced complexes are c Jun c Jun, c Jun JunB, c Jun Fra 1, JunB Fra 1 or JunB JunB, while only the latter two would be present at later stages. DCA enhances the basal expression levels of Fra 1, JunB and c Jun proteins Induction of AP 1 DNA binding activity can be achieved by activation of pre existing Fos Jun proteins or through induction of de novo protein expression.

To differen tiate between these two possibilities, the protein expres sion levels of these molecules was assessed by Western blotting in SKGT4 cells following DCA treatment for 1 6 hr. SKGT4 cells express basal levels of Fra 1, JunB and c Jun. The expression levels of all three proteins are further enhanced Cilengitide by DCA treatment.