Table 1. Antibodies used for chromatin immunoprecipitation (ChIP) Protein Antibody Manufacturer References Western blot ChIP p65 sc-372 Santa Cruz (1, 2) (3-5) Biotechnology p50 sc-1190 Santa Cruz (6) (7) Biotechnology RelB sc-226 Santa Cruz (8) (5) Biotechnology c-Rel sc-71 Santa Cruz (8) (5, 9) Biotechnology p52 06-413 Upstate Biotechnology, (10) Lake Placid, NY E2F4 sc-1082 Santa Cruz (11) (12) Biotechnology Pol II 8WG16 (13) (14) The fact that the p65 dataset has no targets in unstimulated cells confirms the lack of background enrichment from the Dynal magnetic beads. The number of p50 targets in unstimulated cells is higher than for the other subunits. We were concerned that this might be due to the antibody used. Therefore, we tested another antibody raised against the N-terminus of the protein (sc-1191). In test experiments, 90% of enriched gene promoters were shared between the two p50 antibodies (data not shown). 1. Rodriguez, M. S., Thompson, J., Hay, R. T. & Dargemont, C. (1999) J. Biol. Chem. 274, 9108-9115. 2. Huang, T. T., Kudo, N., Yoshida, M. & Miyamoto, S. (2000) Proc. Natl. Acad. Sci. USA 97, 1014-1019. 3. Nissen, R. M. & Yamamoto, K. R. (2000) Genes Dev. 14, 2314-2329. 4. Martone, R., Euskirchen, G., Bertone, P., Hartman, S., Royce, T. E., Luscombe, N. M., Rinn, J. L., Nelson, F. K., Miller, P., Gerstein, M., et al. (2003) Proc. Natl. Acad. Sci. USA 100, 12247-12252. 5. Saccani, S., Pantano, S. & Natoli, G. (2003) Mol. Cell 11, 1563-1574. 6. Yamazaki, T. & Kurosaki, T. (2003) Nat. Immunol 4, 780-786. 7. Baek, S. H., Ohgi, K. A., Rose, D. W., Koo, E. H., Glass, C. K. & Rosenfeld, M. G. (2002) Cell 110, 55-67. 8. Wang, W., Tam, W. F., Hughes, C. C., Rath, S. & Sen, R. (1997) Immunity 6, 165-174. 9. Liu, J. & Beller, D. I. (2003) J. Immunol. 170, 4489-4496. 10. Senftleben, U., Cao, Y., Xiao, G., Greten, F. R., Krahn, G., Bonizzi, G., Chen, Y., Hu, Y., Fong, A., Sun, S. C. & Karin, M. (2001) Science 293, 1495-1499. 11. Takahashi, Y., Rayman, J. B. & Dynlacht, B. D. (2000) Genes Dev. 14, 804-816. 12. Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A. & Dynlacht, B. D. (2002) Genes Dev. 16, 245-256. 13. Hirakata, M., Okano, Y., Pati, U., Suwa, A., Medsger, T. A., Jr., Hardin, J. A. & Craft, J. (1993) J. Clin. Invest. 91, 2665-2672. 14. Odom, D. T., Zizlsperger, N., Gordon, D. B., Bell, G. W., Rinaldi, N. J., Murray, H. L., Volkert, T. L., Schreiber, J., Rolfe, P. A., Gifford, D. K., et al. (2004) Science 303, 1378- 1381. Table 2. Gene ontology (GO) categories enriched in the set of genes bound by NF-κB GO system GO category Number of Total number P value bound genes of genes in category Biological process Response to biotic stimulus 44 516 6.3E-07 Biological process Immune response 38 425 1.3E-06 Biological process Defense response 39 475 7.8E-06 Biological process Response to pest/pathogen/parasite 26 276 3E-05 Molecular function Cytokine activity 15 133 0.00011 Molecular function Transcription factor activity 32 468 0.00046 Biological process Response to stress 33 465 0.00068 Biological process Response to wounding 16 163 0.0007 Molecular function Tumor necrosis factor receptor binding 4 12 0.00072 Biological process Response to external stimulus 49 793 0.00083 Molecular function Transcription regulator activity 38 635 0.00172 Biological process Antigen processing, endogenous 3 7 0.00193 antigen via MHC class I Biological process Inflammatory response 12 116 0.00207 Molecular function Receptor binding 23 332 0.00255 Biological process Antigen processing 3 8 0.003 Biological process Response to stimulus 53 934 0.00345 Biological process Transcription 50 869 0.00347 Biological process Innate immune response 12 125 0.00387 Biological process Regulation of transcription from Pol II 13 143 0.0043 promoter Biological process Response to virus 5 28 0.00442 Molecular function GTP binding 12 139 0.00517 Molecular function Chemokine receptor binding 5 32 0.00592 Molecular function Chemokine activity 5 32 0.00592 Molecular function Guanyl nucleotide binding 12 143 0.00648 Molecular function G-protein-coupled receptor binding 5 33 0.00677 Biological process Transcription, DNA-dependent 47 833 0.00681 Biological process Cell motility 14 169 0.00707 Biological process Viral life cycle 4 20 0.00714 Molecular function DNA binding 48 930 0.00858 Biological process Transcription from Pol II promoter 20 285 0.00902 Molecular function Binding 149 3,561 0.0091 Fisher exact scores were generated using the EASEonline tool (aaps1.niaid.nih.gov/david). A total of 5,707 genes present of the arrays were annotated by GO biological process information; 227 of these were bound by NF-κB. Table 3. Genes bound by NF-κB in U937 cells EntrezGene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52 ABHD8 hypothetical protein FLJ11743 + ACTR3 ARP3 actin-related protein 3 homolog + + AF093680 similar to mouse Glt3 + w AF15Q14 AF15q14 protein + w AIM2 absent in melanoma 2 + + + + + ALAS1 aminolevulinate, delta-, synthase 1 + + + + + AP1S2 adaptor-related protein complex 1 + w + + APBB1IP similar to proline-rich protein 48 + + + + + APG-1 heat shock protein (hsp110 family) w + AQP9 aquaporin 9 + + + + + ARF3 ADP-ribosylation factor 3 + + + + ARHGAP4 Rho GTPase activating protein 4 + w ARHGEF2 rho/rac guanine nucleotide exchange factor + + + + + + ARL1 ADP-ribosylation factor-like 1 + + + X ARL6IP5 vitamin A responsive; cytoskeleton related + + w + w ARTS-1 type 1 TNFR shedding aminopeptidase regulator + + ASGR2 asialoglycoprotein receptor 2 + ASH1 hypothetical protein ASH1 + + + + ATF6 activating transcription factor 6 + w ATP5G1 ATP synthase, H+ transporting + w BAT3 HLA-B associated transcript 3 w + BAZ1B bromodomain adjacent to zinc finger domain, 1B + w BCL2A1 BCL2-related protein A1 + + + + w X BCL6 B-cell CLL/lymphoma 6 (zinc finger protein 51) + w BF B-factor, properdin + w X BNIP1 BCL2/adenovirus E1B 19kD interacting protein 1 + + + BPI bactericidal/permeability-increasing protein + C10orf117 AD24 protein + C11orf10 chromosome 11 open reading frame 10 + w C14orf111 CGI-35 protein + + C20orf28 chromosome 20 open reading frame 28 + C21orf45 chromosome 21 open reading frame 45 + + C21orf59 chromosome 21 open reading frame 59 w + + C3 complement component 3 + + + + w + X C6orf109 DKFZP566C243 protein + + C6orf66 HSPC125 protein + w + + C7orf10 chromosome 7 open reading frame 10 + + + C9orf100 hypothetical protein FLJ14642 + w + + CA11 carbonic anhydrase XI + + + + CAPZA1 capping protein (actin filament) muscle Z-line + CARD15 caspase recruitment domain family, member 15 + X CAT catalase + + CCDC2 capillary morphogenesis protein 1 + CCL1 small inducible cytokine A1, I-309 + + + + X CCL3 small inducible cytokine A3, Mip-1-alpha + + + + + + + + X CCL4 small inducible cytokine A4, Mip-1-beta + + + + w + X CCL5 small inducible cytokine A5 (RANTES) w w + + + X CCNL1 cyclin L ania-6a + + + + + + + + CCR1 chemokine (C-C motif) receptor 1 + w CCRL2 chemokine (C-C motif) receptor-like 2 + + + + w CD37 CD37 antigen w + CD58 CD58 antigen, (LFA 3) w + + CD69 CD69 antigen (p60, early T-cell activation antigen) w + + + + X CDCA1 cell division cycle associated 1 + w w CDH15 cadherin 15, M-cadherin (myotubule) + + CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) + X CHI3L2 chitinase 3-like 2 w + + + CLCN1 chloride channel 1, skeletal muscle (Thomsen disease) w + CLECSF14 macrophage lectin 2 (calcium dependent) + + w COX4I1 cytochrome c oxidase subunit IV isoform 1 + + CREB1 cAMP responsive element binding protein 1 + + + + + + + CRYBA1 crystallin, beta A1 + CSF2 colony stimulating factor 2 (granulocyte-macrophage) + + + + + X CSF3 colony stimulating factor 3 (granulocyte) + + w X CST7 cystatin F (leukocystatin) + w CTSC cathepsin C + + CYB5 cytochrome b-5 + w CYP51A1 cytochrome P450, 51 (lanosterol 14-alpha-demethylase) + DAF decay accelerating factor for complement + + + + + + DBI diazepam binding inhibitor (GABA receptor modulator) + + + + + DBP D site of albumin promoter binding protein + DDX21 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 21 + DDX31 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 31 + DDX47 hqp0256 protein w + + + DGCR6L DiGeorge syndrome critical region gene 6 like + DGUOK deoxyguanosine kinase + DJ434O14.3 hypothetical protein dJ434O14.3 + DKFZp434K1210 hypothetical protein DKFZp434K1210 + + + + DKFZp762L0311 hypothetical protein DKFZp762L0311 + + + Entrez Gene Description + LPS - LPS Known targets symbol p65 c-Rel RelB p50 p52 p65 c-Rel RelB p50 p52 DMAP1 DNA methyltransferase 1-associated protein 1 w + + DPP3 dipeptidylpeptidase III w + + DR1 down-regulator of transcription 1, TBP-binding + + DXS9879E DNA segment on chromosome X, expressed sequence + + EFEMP2 EGF-containing fibulin-like extracellular matrix protein 2 w + EFNB1 ephrin-B1 + + EIF2S3 eukaryotic translation initiation factor 2, subunit 3 w + + + + + ELF3 E74-like factor 3 (ets domain transcription factor) + X ELK1 ELK1, member of ETS oncogene family + EMP3 epithelial membrane protein 3 + + + + ERBB2IP erbb2 interacting protein + + + w ERCC1 excision repair cross-complementing repair deficiency + ET hypothetical protein ET w + EXTL2 exostoses (multiple)-like 2 + + FBXW3 F-box and WD-40 domain protein 3 + X FCAR Fc fragment of IgA, receptor for + + + + FEN1 flap structure-specific endonuclease 1 + + + + FHL3 four and a half LIM domains 3 + + w + w FIBP fibroblast growth factor (acidic) intracellular binding protein + + FKSG87 FKSG87 protein + + + FLJ10420 hypothetical protein FLJ10420 + + + + w FLJ10687 hypothetical