FIGURE 8.

The mechanisms of the regulation of VSNL1 transcription. A, EMSA analysis of the effect of CpG methylation on binding of NRF-1 to its binding site. 5 μg of nuclear proteins extracted from NCI-H520 cells were incubated with ³²P-labeled NRF-1, NRF-1 M1, or NRF-1 M2 oligonucleotides. 60× unlabeled oligonucleotides were used as competitors and incubated with nuclear extract and binding buffer for 30 min before the addition of the ³²P-labeled probe. For supershift, NRF-1 antibody was incubated with the reaction mixture before the addition of the ³²P-labeled probe. B, competing of NRF-1 binding by NRF-1 M1 or NRF-1 M2 oligonucleotides. Nuclear proteins extracted from NCI-H520 cells were incubated with ³²P-labeled NRF-1 oligonucleotides. 60× unlabeled NRF-1, NRF-1 M1, or NRF-1 M2 oligonucleotides were used as competitors. C, the interaction of MBP and HDAC proteins with VSNL1 promoter was analyzed by ChIP assay.