Nuclear Extraction and DNA-Protein Binding Assays

Nuclear extracts were prepared from the various cell lines using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich, St. Louis, MO). Protein concentration was measured with a Bio-Rad protein assay (Hercules, CA), and samples were stored at −70°C until use. Double-stranded DNA oligonucleotide probes corresponding to the predicted TF binding sites of KIR ProI region were synthesized (Figures 2, 3, 5). Labeling and DNA-protein binding reactions were performed as previous described^{9 (link)}. For antibody supershift experiments, nuclear extracts were incubated with 2 µL of antibody for 1 h on ice before the addition of ³²P-labeled DNA probe. After the addition of labeled DNA probe, the binding reaction was incubated for an additional 20 min at room temperature. The antibodies used were cFos (6-2H-2F), FosB (102), cJun (H-79), JunB (C-11), JunD (329), Fra-2 (Q-20), ATF-1 (FI-1), Ets-1 (C-4), Elf-1 (C-20), Oct-1 (E-8, C-21 and 12F11), Oct-3/4 (C-10), Oct-2 (C-20), CREB-1 (24H4B), C/EBPα (D-5), C/EBPβ (H-7), C/EBPγ (H-50), SP-1 (E-3), IRF-1 (C-20), IRF-3 (SL-12), ICSBP (C-19), PU.1 (A-7), E4BP4 (B-1), and MyoD (C-20) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) at gel shift grade (200 µg/0.1µl)