DNA binding proteins that bind to the −80/−17 nt region of the RAG-2 promoter in B-lineage cells.
(A) Nuclear proteins binding to RAG-2 promoter. EMSA was performed as described in “Materials and methods” by incubating nuclear extracts prepared from 18.8.1 cells, L cells, or NIH3T3 cells and the32P-labeled probe DNA (the −80/−17 nt region of the RAG-2 promoter in Figure 1B) in the absence or presence of 100-fold the excess amount of cold probe DNA. C1 and C2 indicate complexes of nuclear protein and probe DNA, and F indicates free probe DNA. (B) Analysis of the R2BP binding region. EMSA was performed using nuclear extracts prepared from 18.8.1 cells and the32P-labeled probe DNA as mentioned above in the presence of various competitor DNA (A-F), as depicted on the top. (C) Mutation analysis of the binding region of R2BP. The RAG-2 promoter fragment (−80/−17 nt region) containing altered nucleotides was prepared (M1-M4, shown on the top). EMSA was performed as in panel B in the presence of 50-fold and 200-fold molar excess of the wild type −80/−17 fragment and the M1, M2, M3, or M4 fragment. Histograms indicate the relative density of the C2 complex shown on the bottom. The density of the C2 complex, in the absence of a competitor, is denoted as 100%.
