ER71 directly interacts with OVOL2. (A) A schematic diagram of OVOL2 and its deletion mutants. (B) GST-ER71 interacts with OVOL2. Binding between recombinant GST-ER71 and OVOL2 from D3.5 EB was determined by immunoblotting by anti-OVOL2 antibody. (C) Coimmunoprecipitation between ER71 and OVOL2. Cell lysates of 293T cells that were transfected with the control or expression plasmid of ER71-MYC, ETS1-MYC, ETS2-MYC, and/or HA-OVOL2 were immunoprecipitated (IP) with anti-MYC antibodies and immunoblotted with anti-MYC and anti-HA antibodies, respectively. β-ACTIN was used for loading control. (D) iFLAG-ER71-HA-OVOL2 ESCs in which the expression of ER71 and OVOL2 is dependent upon doxycycline (DOX) were differentiated in a serum-containing media, treated with DOX at D1 and harvested at D3.5 of differentiation. Subsequently, cell lysate was subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-FLAG antibodies. β-ACTIN was used for loading control. (E) Colocalization of ER71 and OVOL2; 293T cells transiently transfected with ER71-MYC and/or HA-OVOL2 were immunostained with antibodies against MYC or HA, and then analyzed by confocal microscopy. Red and green denote ER71 and OVOL2, respectively. DAPI was used for visualization of nucleus (blue), and all fluorescent images were overlaid with phase contrast images (Merge, last column of panels). All images are magnified views of the original images in supplemental Figure 2. Scale bars, 10 μm. (F) GST-ER71 pull-down with in vitro translated OVOL2. Purified GST-ER71 was incubated with each ³⁵S-labeled WT or mutants OVOL2. Subsequently, pull-down products were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by analysis of radioactive signals by phosphor imager. The phosphor image of input amount of mutant OVOL2 given for binding (top); the phosphor image of mutant OVOL2 that was precipitated by GST-ER71 (middle), and Coomassie blue staining for GST-mER71 that was used for each binding (bottom). (G) Quantification of radioactive signals from in vitro pull-down assays. Binding affinity between GST-ER71 and ³⁵S-labeled OVOL2 mutants were analyzed and presented in the graph. The density of ³⁵S-labeled OVOL2 was normalized against that of GST-ER71, after which it was renormalized against OVOL2 input signal. Value from WT OVOL2 was given at 100%. Results are mean ± standard error of the mean (SEM) from 3 replications. (*P < .05 and ***P < .001 compared with WT). (H-I) Peptide competition assay. (H) The phosphor image of ³⁵S-labeled OVOL2 precipitated with GST-ER71 in the absence or presence of ZF peptide. (I) Quantification of radioactive signals from peptide competition assays. Binding affinity between GST-ER71 and ³⁵S-labeled OVOL2 were analyzed. The density of OVOL2 was normalized against that of GST-ER71. Results are mean ± SEM from 3 replications (***P < .001 compared with no peptide control).