Expression of recombinant OAZ, EHZF, and their N-terminal domains in 293 cells. (A) Nuclear extracts were prepared from 293 cells 48 hours after transfection with either p3xFlagCMV7.1 void vector (F), pFlagCS2-OAZ(KIAA0760-L), pFlagCS2-OAZ(Zf1-13), p3xFlagCMV7.1-EHZF, or p3xFlagCMV7.1-EHZF-N-terminal (1-2000 bp). The extracts were analyzed by Western blotting with M2 antiflag antibody followed by antimouse antibody conjugated to horseradish peroxidase and ECL. (B) Lack of EHZF binding to the X2.RBS oligonucleotide. The nuclear extracts were analyzed by EMSA using 32P-labeled X2.RBS oligonucleotide. In each, panel F indicates free X2.RBS (no nuclear extract added); +, none; nuclear extract, no competition; +AP1, competition with 10-fold molar excess of unlabeled AP1 oligonucleotide; +X2M, competition with 10-fold molar excess of unlabeled X2.Mut oligonucleotide; +X2, competition with 10-fold molar excess of unlabeled X2.RBS oligonucleotide; +αFL, supershift with M2 anti–flag antibody. The higher molecular-weight complex in the 1 to 13 lanes is likely to represent 1 to 13 aggregates. The shift present in the uncompeted EHZF and EHZF-N lanes presumably reflects aspecific binding, since it is abolished by all specific and aspecific competitors. (C) Biotinylated oligonucleotide precipitation assay for the detection of BRE-interacting proteins. 293 cells were transfected with the pFlagCS2-OAZ(KIAA0760-L) or p3xFlagCMV7.1-EHZF vectors and cotransfected, when indicated, with pCDNA3-Smad1-His tagged and pCDNA-Smad4-His. The cells were treated with BMP4 (25 ng/mL) in the presence of 0.5% serum for 16 hours, 36 hours after transfection. Cell extracts were prepared, incubated with BRE-biotinylated oligonucleotide, and streptavidin-magnetic beads were used to “pull down” the proteins associated to the oligonucleotide. Western blotting was performed to identify input and “pulled down” proteins with an anti–his antibody (1:100) recognizing the Smad tagged-his constructs, and the anti–flag M2 antibody (1:500), which binds the Flag-OAZ and Flag-EHZF proteins. The figure shown is representative of 3 independent “pull down” experiments.
