Figure 6

Detection of DNA-protein interactions using electrophoretic mobility shift/supershift assays in vitro of consensus binding sequences in the 5'promoter of MTG16 and nuclear extracts. The following sequence of the oligonucleotide probe was used in EMSA for the conserved GATA (-301) core consensus site (red): GATA-301 probe 5'- CCCGGCATTATCACGGGGACAC. Results are given for HEL/MEG-01/TF-1/UT-7 cells. Primary DNA-nuclear protein interactions are shown by arrows marked shift; DNA- nuclear protein-antibody interactions are shown by arrows marked supershift. All cell lines tested showed identical. A shift is shown for the GATA -301 probe that is competed for by excess unlabelled probe (competitor) (3) indicating binding of nuclear extract protein to the biotinylated probe that containing the the GATA -301 sequence. Proteins bound to the probe were "super-shifted" by antibody to GATA-1 (4) but not with antibody to GATA-2 (5). All experiments were repeated at least twice.