DNA binding of renatured nuclear proteins fractionated by SDS-PAGE. U937 nuclear proteins were separated by SDS-PAGE. The gel was cut crosswise in fractions on the basis of molecular weight, and the nuclear proteins were eluted and renatured. (A) Distribution of DNA-binding activity in gel size-fractionated nuclear proteins. Renatured proteins (10 μL) from different molecular weight fractions were analyzed by EMSA using the labeled FCAR Ets oligonucleotide (wtETS). The molecular weights indicated correspond to molecular weight size markers (Prestained SDS-PAGE Standard; Bio-Rad, Hercules, CA) run alongside the nuclear extract. U937, one third of the binding reaction with 6 μg U937 nuclear extract was applied to the electrophoresis. ns indicates nonspecific bands identified previously.²⁷  (B) DNA-binding specificity and supershift EMSA of size-fractionated nuclear factors that bind to the FCAR Ets site. Renatured proteins (6 μL) from fraction 12 were analyzed by EMSA using the labeled FCAR Ets oligonucleotide (wtETS) in the absence (lane 1) or presence of 100-fold molar excess of unlabeled wild-type (lane 2) and mutant FCAR Ets oligonucleotide competitors (mutETS) (lane 3), or 2 μg antibody against Elf-1 (lane 4). Arrow indicates the binding complex comigrating with HEL-NF1. The supershifted band is indicated by open circle. (C) DNA binding of gel size-fractionated nuclear proteins in the presence of the GABPβ subunit. The indicated SDS-PAGE fractions (10 μL) were incubated with the labeled wtETS-s oligonucleotide in the absence (-) or presence of 1 μL in vitro-translated GABPβ (+). U937, one third of the binding reaction with 6 μg U937 nuclear extract was applied to the electrophoresis.