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Fig. 1. EGR-4 and EGR-3 bind NF-B p50 and p65 proteins. (A) Recombinant p50 and p65 expressed in E. coli as fusion proteins were coupled to GST matrix, and cell extract containing recombinant or Jurkat-cell-derived native EGR-4 or EGR-3 was applied to this matrix. After loading, the columns were extensively washed and bound proteins were eluted with glutathione. Cell lysates (Ly) and eluates (el) were separated by SDS-PAGE and analysed by western blotting. Use of surface-plasmon-resonance assays to identify binding of NF-B p50 (B) and p65 (C) to immobilized EGR-4. NF-B p50 and p65 were injected into a flow cell precoupled with recombinant EGR-4. The control values obtained by measuring absorbance of NF-B proteins to an uncoupled chip were subtracted from the profile with immobilized proteins. Representative experiments are shown. (D) Jurkat-cell extract was used to immunoprecipitate EGR-4 using p65 antiserum. The fractions (flow through, wash and eluate) were separated by SDS-PAGE and used for western blotting. p65 and EGR-4 were detected in the fraction obtained after loading the column (lane 2), the first wash fraction (lane 3) and in the eluate (lane 5). Cell extract with recombinant EGR-4 or p65 proteins are shown in lane 1.

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