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. 2009 Nov 12;285(2):903–913. doi: 10.1074/jbc.M109.075630

FIGURE 3.

FIGURE 3.

A, c-Jun enhances PDK1 transactivation through Sp1 and Ets. c-Jun interacts with Sp1 in melanoma cells. Nuclear extracts from MeWo cells and MeWo cells stably transfected with FLAG-c-Jun were immunoprecipitated (IP) with FLAG antibodies and blotted (IB) with the indicated antibodies. B, chromatin immunoprecipitation assay. The products of PDK1- and glyceraldehyde-3-phosphate dehydrogenase-specific PCR amplification on IP material from Lu1205 cells are shown. c-Jun antibodies sc-44 and sc-45 were used. C, c-Jun siRNA decrease binding of c-Jun to the PDK1 promoter. A chromatin immunoprecipitation assay was performed with extracts from Lu1205 cells transfected with scramble (sc) or c-Jun siRNA. D, protein extracts from cells described in panel C were analyzed by Western blot using the indicated antibodies. E and F, c-Jun binds to the box 3 region from the PDK1 promoter. Protein extracts from c-Jun−/− and c-Jun+/+ mouse embryonic fibroblasts (MEFs) and Lu1205 cells were incubated with a biotinylated oligo bearing the box 3 sequence. Complexes were pulled down with streptavidin and blotted with the indicated antibodies. G, Ets-1 is involved regulation of PDK1 expression. Protein extracts obtained from Lu1205 cells transfected with scramble (sc) or Ets-1 siRNA were analyzed by Western blot using the indicated antibodies. H, c-Jun regulates Ets-1 expression. Protein extracts from Lu1205 transduced with c-Jun lentiviral shRNA were analyzed by Western blot using the indicated antibodies. PCNA, proliferating cell nuclear antigen.