Figure 4

In vitro binding of NFI-A to its full binding site in the mouse L1 gene is confirmed by supershift and competition assays. EMSAs were performed with ³²P-labeled oligonucleotides indicated above the autoradiograms (for a description, see Fig. 3). In both images (A-B), only the upper and lower parts of the respective autoradiogram are shown, the middle sections, which did not exhibit any signals, were cut out for reasons of space. A, Supershift experiment. NFI-A st "+": oligonucleotides were incubated with extracts from CHO cells transfected with pCHNFI-A (NFI-A st); NFI-A st "-": oligonucleotides were not pre-incubated with cell extracts (negative control). Where indicated, anti-HA antibody was added. Arrow a points at anti-HA-antibody/NFI-A/DNA super-shifted complexes, while arrow b indicates NFI-A/DNA complexes. B, Competition analysis with the radioactively labeled NFI binding site from the mouse L1 gene regulatory region (L) and various unlabeled competitor oligonucleotides (N, Nc, L, Lc; see Fig. 3 for a description). The ³²P-labeled L oligonucleotide was incubated with extracts from CHO cells transfected with pCHNFI-A (NFI-A st) in the presence of different molar excesses of the unlabeled competitor oligonucleotides as indicated above the autoradiogram. A "-" indicates that oligonucleotides were not pre-incubated with cell extracts (negative control). The arrow points at the main band representing NFI-A-DNA complexes.