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Figure 5 | Molecular Cancer

Figure 5

From: Catecholamine up-regulates MMP-7 expression by activating AP-1 and STAT3 in gastric cancer

Figure 5

c-Jun and STAT3 synergistically regulate MMP-7 expression. A and B, MGC-803 cells were stimulated with 10 μM of isoproterenol for 0, 2.5 or 4 h, respectively. The chromatin DNA/nuclear protein complexes were prepared. Binding of STAT3 and c-Jun to the AP-1 site was analyzed by the immunoprecipitation with anti-STAT3 and anti-AP-1 antibodies followed by PCR (A) and real-time PCR (B). C, MGC-803 cells were treated with 10 μM isoproterenol for 0 or 2.5 h, respectively. The nuclear extract was prepared with a Nuclear-Cytosol Extraction Kit. β-tubulin and AP-2α were analyzed by Western blot to examine the separation of cytoplasmic and nuclear proteins. C, cytosolic proteins; N, nuclear proteins. D, 200 μg of the nuclear extracts was incubated at 4°C for 4 h with the biotinylated oligonucleotides containing AP-1 consensus sequence previously coupled to Dynabeads M-280 in the presence or absence of one, five or 15 fold amount of the double-stranded oligonucleotide competitors. Incubation of the nuclear proteins with the double-stranded oligonucleotides containing a mutated AP-1 site (Mut Oligo) or non-specific double-stranded oligonucleotides (NS Oligo) was used as controls. The protein/DNA complexes were separated with a Dynal magnet, and subjected to SDS-PAGE. STAT3 and c-Jun were detected by Western blot with anti-STAT3 and anti-c-Jun antibodies. E and F, MGC-803 cells were co-transfected with the plasmids pMMP-7mA and pCDNA3.1/c-Jun or STAT3C and luciferase activities were assayed.

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