Figure 4.

Egr4 binds the Egr4^KCC2 element in vitro. a, Three specific complexes (I–III) are detected in EMSA, using labeled oligonucleotides bearing a single copy of the Egr4^KCC2 element and nuclear extracts of N2a cells transfected with Egr4 expression plasmid. Complex I is competed effectively by cold Egr4^KCC2 oligonucleotides. In contrast, unlabeled Egr4^KCC2mut oligonucleotides do not influence the formation of complex I. The addition of Egr4 antibody prevents complex I formation in a dose-dependent manner and results in the formation of a new supershifted complex (indicated by ss). Complex II is affected less by Egr4^KCC2 oligonucleotides and not at all by Egr4^KCC2mut oligonucleotides. Complex III is competed by both wild-type and mutant Egr4^KCC2 oligonucleotides. Note the significant increase in complex III intensity after the addition of the Egr4 antibody. Nonspecific binding is indicated by an asterisk. b, Sequences of Egr4^KCC2 and Egr4^KCC2mut oligonucleotides used in EMSA. The Egr4^KCC2 sequence is highlighted in bold. Mutated nucleotides are shown in small case letters.