Figure 3

NFI-A binds to its full binding site in the mouse L1 gene in vitro. ³²P-labeled oligonucleotides used for EMSA: L: full NFI binding site in mouse L1 gene with flanking sequences; Lc: point-mutated variant of L; N: idealized NFI binding site with flanking sequences [18]; Nc: point-mutated variant of N; C: oligonucleotide without NFI recognition motif (SIS oligonucleotide). NFI binding site sequences are capitalized, point mutations are shown in boldface. The sequence of one strand is shown after the fill-in reaction. Oligonucleotides were incubated with extracts from CHO cells expressing NFI-A bs or NFI-A st, or from mock-transfected cells (CMV). Only the upper and lower parts of the autoradiogram are shown, the middle section, which did not exhibit any signals, was cut out for reasons of space. Arrows A: HA-NFI-A-DNA complexes; arrow B: complexes of endogenous NFI and DNA; arrows C and D: free oligonucleotide DNA. Note that that the free SIS oligonucleotide migrates slightly slower (arrow C) than the other free oligonucleotides (arrow D) due to its higher molecular weight.