FIGURE 4.

S489F and M513I substitutions affect Loz1 DNA binding in vitro. A, DNA sequence of the wild-type and mutated oligonucleotide probes. The loz1 binding site is boxed. B, SDS-PAGE analysis of the purified Trx-histidine tag (vector) and recombinant proteins TH-loz1, TH-loz1 C470G, TH-loz1 S489F, TH-loz1 M513I, or TH-loz1 ZF. Proteins were visualized by staining with Coomassie Blue. The sizes of the molecular mass markers are shown in kDa on the left. C, representative EMSA using 40 μmol of ³²P-labeled double-stranded oligonucleotide and 200 ng of the indicated purified recombinant protein. For competition studies, 0, 50×, 200×, or 500× of the WT unlabeled oligonucleotide (Comp Inhibitor), or mutant oligonucleotide (Non-Comp Inhibitor) was added to the reactions.