(A) EMSA of nuclear extracts from 293T cells transfected with FLAG-tagged OCZF cDNA. c-Krox or Egr-1 DNA was incubated with the nuclear extract from 293T cells transfected with FLAG-OCZF cDNA (O; lanes 2 through 5 and 7 through 10) or vector cDNA (v; lanes 1 and 6), respectively. *The complex of OCZF and DNA. Competition experiments were performed in the presence of 100-fold molar excess of unlabeled c-Krox (lane 3), Egr-1 (lane 8), or SP1 (lanes 4 and 9) DNA. Binding reactions were also performed after preincubation of nuclear extracts with anti-FLAG MoAb (lanes 5 and 10). Thirty micrograms of nuclear extracts was analyzed. (B) Analysis of DNA binding activity with S-tagged Egr-1 and OCZF proteins. S-protein pulldown experiments were performed as described in Materials and Methods. The bound DNA was analyzed by a 10% PAGE. Samples purified on S-protein beads were prepared from 293T cells transfected with pCDL-FLAGa (v1; lane 1), pCDL-FLAGc (v2; lanes 3 and 5) or pCDL-FLAG containing Egr-1 (lane 2) or OCZF (lanes 4 and 6). They were analyzed for DNA binding activity using Egr-1 (lanes 1 through 4) or c-Krox (lanes 5 and 6) consensus sequences as probes. Arrows indicate the bound Egr-1 or c-Krox DNA. Lower bands indicate degraded products of Egr-1 DNA. Autoradiography was performed for 6 days at −80°C for OCZF protein or 3 days at room temperature for Egr-1 protein.