Figure 6.

Interaction of TFPI-2 with AP-2α reduces the transcriptional activity of the MMP2 promoter. (A) Protein A beads conjugated with TFPI-2 antibodies or normal IgG were used for Co-IP experiments to analyze the interaction of TFPI-2 with putative transcriptional factors in the nuclear extracts of MDA231/TFPI-2 cells. The precipitates were subjected to western blots probing by anti-TFPI-2, anti-PEA3, anti-AP-2α and anti-SP1 antibodies. The samples are derived from the same experiment and that blots were processed in parallel. (B) Protein A beads conjugated with AP-2α antibodies or normal IgG were used for the reciprocal Co-IP experiments to detect the association of AP-2α with TFPI-2 in the nuclear extracts. The precipitates were subjected to western blot analyses. (C) Western blots show the levels of AP-2α in MDA231 cell lines with or without TFPI-2 overexpression. GAPDH was used as a loading control. (D) Co-IP using AP-2α antibodies confirmed association of TFPI-2 with AP-2α (E) Left: western blots show the down-regulation of AP-2α in MDA231 cells. Right: qRT-PCR indicates that knockdown of AP-2α decreases the expression of MMP-2 mRNA in MDA231 cells. ** < 0.01 as determined by Student’s t-test. (F) ChIP assays were performed to detect the binding ability of AP-2α to the MMP2 gene promoter. Upper panel: schematic presentation of the MMP-2 gene promoter and the two regions to be analyzed by qPCR. The R1 region contains the putative binding sites for AP-2α. Arrows represent primers used for qPCR analysis. Amplicon lengths for R1 and R2 are about 300 bp, respectively. Lower panel: Protein A beads conjugated with Histone 2 (H2), AP-2α or TFPI-2 antibodies were used for ChIP assays. After immunoprecipitation, amplicons corresponding to R1 and R2 were amplified and analyzed. Relative levels of the amplicons are statistically analyzed and the data is presented as means ± SD from three independent experiments. *P < 0.05 as determined by Student’s t-test.