Download Table 1. Antibodies used for chromatin immunoprecipitation (ChIP

Table 1. Antibodies used for chromatin immunoprecipitation (ChIP)
Protein
Antibody
Manufacturer
p65
sc-372
p50
sc-1190
RelB
sc-226
c-Rel
sc-71
p52
06-413
E2F4
sc-1082
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Upstate Biotechnology,
Lake Placid, NY
Santa Cruz
Biotechnology
Pol II
8WG16
References
Western blot
ChIP
(1, 2)
(3-5)
(6)
(7)
(8)
(5)
(8)
(5, 9)
(10)
(11)
(12)
(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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Rodriguez, M. S., Thompson, J., Hay, R. T. & Dargemont, C. (1999) J. Biol. Chem. 274,
9108-9115.
Huang, T. T., Kudo, N., Yoshida, M. & Miyamoto, S. (2000) Proc. Natl. Acad. Sci. USA
97, 1014-1019.
Nissen, R. M. & Yamamoto, K. R. (2000) Genes Dev. 14, 2314-2329.
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.
Saccani, S., Pantano, S. & Natoli, G. (2003) Mol. Cell 11, 1563-1574.
Yamazaki, T. & Kurosaki, T. (2003) Nat. Immunol 4, 780-786.
Baek, S. H., Ohgi, K. A., Rose, D. W., Koo, E. H., Glass, C. K. & Rosenfeld, M. G. (2002)
Cell 110, 55-67.
Wang, W., Tam, W. F., Hughes, C. C., Rath, S. & Sen, R. (1997) Immunity 6, 165-174.
Liu, J. & Beller, D. I. (2003) J. Immunol. 170, 4489-4496.
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.
Takahashi, Y., Rayman, J. B. & Dynlacht, B. D. (2000) Genes Dev. 14, 804-816.
Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R. A. & Dynlacht, B. D.
(2002) Genes Dev. 16, 245-256.
Hirakata, M., Okano, Y., Pati, U., Suwa, A., Medsger, T. A., Jr., Hardin, J. A. & Craft, J.
(1993) J. Clin. Invest. 91, 2665-2672.
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, 13781381.
Table 2. Gene ontology (GO) categories enriched in the set of genes bound by NF-κB
GO system
GO category
Biological process
Response to biotic stimulus
Biological process
Immune response
Biological process
Number of
bound genes
P value
44
Total number
of genes in
category
516
38
425
1.3E-06
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
3
7
0.00193
Biological process
Antigen processing, endogenous
antigen via MHC class I
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
13
143
0.0043
Biological process
Regulation of transcription from Pol II
promoter
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
6.3E-07
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
ABHD8
hypothetical protein FLJ11743
ACTR3
ARP3 actin-related protein 3 homolog
AF093680
similar to mouse Glt3
AF15Q14
AF15q14 protein
AIM2
absent in melanoma 2
ALAS1
aminolevulinate, delta-, synthase 1
AP1S2
adaptor-related protein complex 1
APBB1IP
similar to proline-rich protein 48
APG-1
heat shock protein (hsp110 family)
AQP9
aquaporin 9
ARF3
ADP-ribosylation factor 3
ARHGAP4
Rho GTPase activating protein 4
ARHGEF2
rho/rac guanine nucleotide exchange factor
ARL1
ADP-ribosylation factor-like 1
ARL6IP5
vitamin A responsive; cytoskeleton related
ARTS-1
type 1 TNFR shedding aminopeptidase regulator
ASGR2
asialoglycoprotein receptor 2
ASH1
hypothetical protein ASH1
ATF6
activating transcription factor 6
ATP5G1
ATP synthase, H+ transporting
BAT3
HLA-B associated transcript 3
BAZ1B
bromodomain adjacent to zinc finger domain, 1B
BCL2A1
BCL2-related protein A1
BCL6
B-cell CLL/lymphoma 6 (zinc finger protein 51)
BF
B-factor, properdin
BNIP1
BCL2/adenovirus E1B 19kD interacting protein 1
BPI
bactericidal/permeability-increasing protein
C10orf117
AD24 protein
C11orf10
chromosome 11 open reading frame 10
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
C3
complement component 3
C6orf109
DKFZP566C243 protein
C6orf66
HSPC125 protein
C7orf10
chromosome 7 open reading frame 10
C9orf100
hypothetical protein FLJ14642
CA11
carbonic anhydrase XI
CAPZA1
capping protein (actin filament) muscle Z-line
CARD15
caspase recruitment domain family, member 15
CAT
catalase
CCDC2
capillary morphogenesis protein 1
CCL1
small inducible cytokine A1, I-309
CCL3
small inducible cytokine A3, Mip-1-alpha
CCL4
small inducible cytokine A4, Mip-1-beta
CCL5
small inducible cytokine A5 (RANTES)
CCNL1
cyclin L ania-6a
CCR1
chemokine (C-C motif) receptor 1
CCRL2
chemokine (C-C motif) receptor-like 2
CD37
CD37 antigen
CD58
CD58 antigen, (LFA 3)
CD69
CD69 antigen (p60, early T-cell activation antigen)
CDCA1
cell division cycle associated 1
CDH15
cadherin 15, M-cadherin (myotubule)
CDKN1A
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
CHI3L2
chitinase 3-like 2
CLCN1
chloride channel 1, skeletal muscle (Thomsen disease)
CLECSF14
macrophage lectin 2 (calcium dependent)
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)
CSF3
colony stimulating factor 3 (granulocyte)
CST7
cystatin F (leukocystatin)
CTSC
cathepsin C
CYB5
cytochrome b-5
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
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
- LPS
+
+
+ w
+ +
+ +
+
+ +
+
+ +
w
+ +
+
+ +
+
++
+w
w
++
++
+
++
w
++
++
+w
++
+
+w
+
+
++
+w
+
+
+
++
+w
w
++
+
+
+
+
+
w+
+ + ++
+
+
+
+
+ w++
+
+
+
+
+
+ ++
+ + ++
+ + ++
w w+
+ + ++
+w
+ + ++
w
w+
w+ ++
+w
+
+
w+
w+
++
+
+ ++
++
++
+
+
+
+
+
+
+
+
+
+
+ w
+
X
w
w
w
+
X
X
w
+
+
+
+
++
+
w
w
+
+
+ ++
+
+
+ ++
+
w
+
+
w
+
+
+
+
w
++
+
+
+
w++
+
+ +
+ +
X
++
+
++ +
+
+w
+ w
+
+
+
+ + ++ +
+
++ +
+
Known targets
p65
c-Rel
RelB
p50
p52
+ LPS
p65
c-Rel
RelB
p50
p52
EntrezGene Description
symbol
+
X
X
X
X
X
X
X
w
+
+ ++
++
+
w
+
+
+
+
+
++
+
X
X
DMAP1
DPP3
DR1
DXS9879E
EFEMP2
EFNB1
EIF2S3
ELF3
ELK1
EMP3
ERBB2IP
ERCC1
ET
EXTL2
FBXW3
FCAR
FEN1
FHL3
FIBP
FKSG87
FLJ10420
FLJ10687
FLJ11082
FLJ11773
FLJ11838
FLJ13195
FLJ13639
FLJ20080
FLJ20257
FLJ20477
FLJ20825
FLJ22353
FLJ22555
FLJ22573
FLOT1
FUT4
GABPB2
GADD45B
GDF5
GNA15
GNG5
GNL1
GPR132
GPR84
GPX1
GTF3C4
GTPBP4
GYPC
HADHA
HCST
HIST1H4I
HIST1H4K
HLA-B
HLA-F
HLA-G
HMGN4
HNMT
HOXA7
HSPA1B
HSPC003
HSPC052
HSPC073
HSPCB
ICAM1
ICAM4
IER3
IFNGR1
IL1B
IL1F9
IL2RG
IL6ST
IL8
ILK
INHBE
ITGA5
ITGAX
ITSN2
IVNS1ABP
JUN
DNA methyltransferase 1-associated protein 1
dipeptidylpeptidase III
down-regulator of transcription 1, TBP-binding
DNA segment on chromosome X, expressed sequence
EGF-containing fibulin-like extracellular matrix protein 2
ephrin-B1
eukaryotic translation initiation factor 2, subunit 3
E74-like factor 3 (ets domain transcription factor)
ELK1, member of ETS oncogene family
epithelial membrane protein 3
erbb2 interacting protein
excision repair cross-complementing repair deficiency
hypothetical protein ET
exostoses (multiple)-like 2
F-box and WD-40 domain protein 3
Fc fragment of IgA, receptor for
flap structure-specific endonuclease 1
four and a half LIM domains 3
fibroblast growth factor (acidic) intracellular binding protein
FKSG87 protein
hypothetical protein FLJ10420
hypothetical protein FLJ10687
hypothetical protein FLJ11082
hypothetical protein FLJ11773
hypothetical protein FLJ11838
hypothetical protein FLJ13195 similar to stromal antigen 3
hypothetical protein FLJ13639
hypothetical protein FLJ20080
hypothetical protein FLJ20257
hypothetical protein FLJ20477
hypothetical protein FLJ20825
hypothetical protein FLJ22353
hypothetical protein FLJ22555
hypothetical protein FLJ22573
flotillin 1
fucosyltransferase 4 (alpha (1,3) fucosyltransferase)
GA binding protein transcription factor, beta subunit 2
growth arrest and DNA-damage-inducible, beta
growth differentiation factor 5
guanine nucleotide binding protein (G protein), alpha 15
guanine nucleotide binding protein (G protein), gamma 5
guanine nucleotide binding protein-like 1
G protein-coupled receptor
G protein-coupled receptor 84
glutathione peroxidase 1
general transcription factor IIIC, polypeptide 4 (90kD)
G protein-binding protein CRFG
glycophorin C (Gerbich blood group)
hydroxyacyl-Coenzyme A dehydrogenase
DNAX-activation protein 10
H4 histone family, member M
H4 histone family, member D
major histocompatibility complex, class I, B
major histocompatibility complex, class I, F
HLA-G histocompatibility antigen, class I, G
high-mobility group protein 17-like 3
histamine N-methyltransferase
homeo box A7
heat shock 70kD protein 1B
HSPC003 protein
HSPC052 protein
HSPC073 protein
heat shock 90kD protein 1, beta
intercellular adhesion molecule 1 (CD54)
intercellular adhesion molecule 4
immediate early response 3
interferon gamma receptor 1
interleukin 1, beta
interleukin-1 homolog 1
interleukin 2 receptor, gamma
interleukin 6 signal transducer (gp130)
interleukin 8
integrin-linked kinase
hypothetical protein MGC4638
integrin, alpha 5 (fibronectin receptor)
integrin, alpha X, alpha polypeptide)
intersectin 2
NS1-binding protein
v-jun sarcoma virus 17 oncogene homolog (avian)
+ LPS
- LPS
Known targets
p65
c-Rel
RelB
p50
p52
p65
c-Rel
RelB
p50
p52
Entrez Gene Description
symbol
+
w+
+
+
w
+
w+ ++
+
+
+ + ++
+ + +w
+
w
+
+
+ + +
+
++
+
+ + +
+ + ++
+
+
+ +
+
+
w +
++
+
+w
+ + ++
+
+
+ + ++
w ++
+ + ++
+ w
+ w++
+ + ++
+
+
+
+
+
+
+
+ +
w
w
+
+
+
w
+ w
w +
w
w
+
+
+
+ + +w
+ + ++
+ + ++
+
+ + w
+
+
+
+
+
++
+ + ++
+w
+ + +w
w
w+
++
+ ++
++
+w
X
+
++
+ w
+
w
+
+
+
w
+
+
++
w
w
+
w ++
w
+
+
++
X
+
+
w
+ + +w
+ + ww
+ + ++
+
+
+ + ++
+
w
+ w+w
+ ++
+ +w
w w+
+
X
w
+
+
w +
+
+
+
+
++
+
+
X
X
+
+
+
+
+
+
+
w
X
X
+ w+
+
w
X
+
w
+
X
X
X
KIAA0186
KIAA0218
KIAA0319
KIAA0409
KIAA0669
KIAA1008
KIAA1056
KLF15
LGALS1
LIM2
LIMK1
LIPT1
LMNA
LOC113201
LOC116211
LOC51015
LOC51321
LOC55580
LOC92106
LRRC19
LRSAM1
M96
MAN2B1
MAPK6
MASTL
MCCC2
MCP
MDH1
MEF2B
MGC10960
MGC10966
MGC14141
MGC4677
MGC4771
MGC5242
MID1IP1
MLLT10
MMAB
MMP19
MMP8
MMP9
MRPL30
MRPL34
MRPS7
MX2
NAG73
NAP1L1
NAPA
NCR1
NFATC2
NFKB2
NFKBIB
NKG7
NME1
NOLC1
NOVA1
NR1D1
NRAS
NRXN3
NUP107
OAS3
OAZ1
OAZ2
ORMDL1
P4HA1
PAFAH2
PAK4
PALM
PDE6D
PEX13
PHLDA1
PILRA
PILRB
PKM2
PLK1
PLXNB1
PMAIP1
PMS1
PMS2L8
KIAA0186 gene product
KIAA0218 gene product
KIAA0319 gene product
KIAA0409 protein
KIAA0669 gene product
mitotic control protein dis3 homolog
KIAA1056 protein
Kruppel-like factor 15
lectin, galactoside-binding, soluble, 1 (galectin 1)
lens intrinsic membrane protein 2 (19kD)
LIM domain kinase 1
lipoyltransferase
lamin A/C
hypothetical protein BC012124
hypothetical protein BC013113
CGI-111 protein
hypothetical protein
hypothetical protein
hypothetical protein BC008322
leucine rich repeat containing 19
leucine rich repeat and sterile alpha motif containing 1
putative DNA binding protein
mannosidase, alpha, class 2B, member 1
mitogen-activated protein kinase 6
hypothetical protein FLJ14813
methylcrotonoyl-Coenzyme A carboxylase 2 (beta)
membrane cofactor protein (CD46)
malate dehydrogenase 1, NAD (soluble)
MADS box transcription factor 2, polypeptide B
hypothetical protein MGC10960
hypothetical protein MGC10966
hypothetical protein MGC14141
hypothetical protein MGC4677
hypothetical protein MGC4771
hypothetical protein MGC5242
hypothetical protein STRAIT11499
myeloid/lymphoid leukemia translocated to 10
hypothetical protein MGC20496
matrix metalloproteinase 19
matrix metalloproteinase 8 (neutrophil collagenase)
matrix metalloproteinase 9 (gelatinase B, 92kD gelatinase)
mitochondrial ribosomal protein L30
mitochondrial ribosomal protein L34
mitochondrial ribosomal protein S7
myxovirus (influenza virus) resistance 2 (mouse)
NPC-related protein NAG73
nucleosome assembly protein 1-like 1
N-ethylmaleimide-sensitive factor attachment protein, alpha
lymphocyte antigen 94 homolog, NK-p46, (mouse)
nuclear factor of activated T-cells, calcineurin-dependent 2
NF-κB p52/p100
NF-κB inhibitor, beta
natural killer cell group 7 sequence
non-metastatic cells 1, protein (NM23A) expressed in
nucleolar and coiled-body phosphprotein 1
neuro-oncological ventral antigen 1
nuclear receptor subfamily 1, group D, member 1
neuroblastoma RAS viral (v-ras) oncogene homolog
neurexin 3
nuclear pore complex protein
2'-5'-oligoadenylate synthetase 3 (100 kD)
ornithine decarboxylase antizyme 1
ornithine decarboxylase antizyme 2
hypothetical protein
procollagen-proline, 2-oxoglutarate 4-dioxygenase alpha
platelet-activating factor acetylhydrolase 2 (40kD)
p21(CDKN1A)-activated kinase 4
paralemmin
phosphodiesterase 6D, cGMP-specific, rod, delta
peroxisome biogenesis factor 13
pleckstrin homology-like domain, family A, member 1
paired immunoglobulin-like receptor alpha
paired immunoglobulin-like receptor beta
pyruvate kinase, muscle
polo-like kinase (Drosophila)
plexin B1
phorbol-12-myristate-13-acetate-induced protein 1
PMS1 postmeiotic segregation increased 1 (S. cerevisiae)
postmeiotic segregation increased 2-like 8
+ LPS
- LPS
p65
c-Rel
RelB
p50
p52
p65
c-Rel
RelB
p50
p52
EntrezGene Description
s ymbol
+
+
+ w
+
+
+
+
++
+
w
++
w
w
+
+
+
++ +
w+
+
+
++
+
+
+ + w
+
w w+
+
+
+ + +
+
+
+ + ++
+ +
++
+
+
w w+
w+ w
w+
w++
+
+ + +w
++
++
+w
+
+
+
++
+
+ ++
+ + +
+
++
+
+
+
+
+
+
+
+ + ++
+ w++
+w
+
+w
Known targets
w
+
+
+
w
+
+ ++
+
w
w
+
+
+
+ +
+
w w
+
+
w
+ +
+
+
+
X
+
+
+
+ ++
w +
+
w+
w+
+
+ w
+ + ++
+
+
+
+ w++ +
w+
w+
+
+w
+
w+
+w
+
+w
w + w
+
w
+
+
++ +
+
+
+
+
+
+
+
+
+
++
w
+
+
+
+
X
w
+
+
+
X
POLB
POLD4
POLR1C
POLR2A
POLR3E
PPFIBP1
PPP1R15B
PPP1R3B
PPP5C
PRDM1
PRDM11
PRDM8
PRKAB2
PTGS2
PTPN7
PTX3
PXMP3
QTRTD1
RAB7L1
RABL2A
RABL2B
RAC2
RB1
RB1CC1
RBBP4
RFP
RFXANK
RGC32
RGL1
RND1
RNF4
RPL27A
RPL39
RPL39L
RPS28
RPS8
RS1
S100A12
S100A3
S100A4
S100A9
S100Z
SAS
SCARB1
SDF2L1
SELPLG
SEMA7A
SEPW1
SERPINB2
SERPINE1
SFRS2
SIRT2
SLAMF8
SLC1A3
SLC25A12
SLC30A7
SLC39A6
SLC7A1
SMPD1
SOX8
SPATA2
SQSTM1
SREBF2
SRF
SSSCA1
STARD10
STAT1
STAT5A
STAT6
STATIP1
STXBP3
SVIL
SYNPO
TAGLN2
TAPBP
TFAP2A
TFDP3
polymerase (DNA directed), beta
polymerase (DNA-directed), delta 4
RNA polymerase I subunit
polymerase (RNA) II (DNA directed) polypeptide A
hypothetical protein FLJ10509
PTPRF interacting protein, binding protein 1
protein phosphatase 1, regulatory subunit 15B
protein phosphatase 1, regulatory subunit 3B
protein phosphatase 5, catalytic subunit
PR domain containing 1, with ZNF domain
PR domain containing 11
PR domain containing 8
protein kinase, AMP-activated, beta 2 subunit
prostaglandin-endoperoxide synthase 2, COX-2
protein tyrosine phosphatase, non-receptor type 7
pentaxin-related gene, rapidly induced by IL-1 beta
peroxisomal membrane protein 3 (Zellweger syndrome)
hypothetical protein FLJ12960
RAB7, member RAS oncogene family-like 1
RAB, member of RAS oncogene family-like 2A
RAB, member of RAS oncogene family-like 2B
ras-related C3 botulinum toxin substrate 2 (rho family)
retinoblastoma 1 (including osteosarcoma)
RB1-inducible coiled-coil 1
retinoblastoma binding protein 4
ret finger protein
regulatory factor X-associated ankyrin-containing protein
RGC32 protein
RalGDS-like gene
GTP-binding protein
ring finger protein 4
ribosomal protein L27a
ribosomal protein L39
ribosomal protein L39-like
ribosomal protein S28
ribosomal protein S8
retinoschisis (X-linked, juvenile) 1
S100 calcium binding protein A12 (calgranulin C)
S100 calcium binding protein A3
S100 calcium binding protein A4 (calvasculin)
S100 calcium binding protein A9 (calgranulin B)
S100Z protein
sarcoma amplified sequence
CD36 antigen (thrombospondin receptor)-like 1
stromal cell-derived factor 2-like 1
selectin P ligand
sema domain, immunoglobulin domain, semaphorin 7A
selenoprotein W, 1
serine (or cysteine) proteinase inhibitor, clade B
serine (or cysteine) proteinase inhibitor, clade E
splicing factor, arginine/serine-rich 2
sirtuin silent mating type information regulation 2
B lymphocyte activator macrophage expressed
solute carrier family 1 (glutamate transporter), member 3
solute carrier family 25, member 12
zinc transporter like 2
LIV-1 protein, estrogen regulated
solute carrier family 7, member 1
sphingomyelin phosphodiesterase 1, acid lysosomal
SRY (sex determining region Y)-box 8
spermatogenesis associated 2
sequestosome 1
sterol regulatory element binding transcription factor 2
serum response factor
Sjogren's syndrome/scleroderma autoantigen 1
serologically defined colon cancer antigen 28
signal transducer and activator of transcription 1
signal transducer and activator of transcription 5A
signal transducer and activator of transcription 6
elongator protein 2
syntaxin binding protein 3
supervillin
synaptopodin
transgelin 2
TAP binding protein (tapasin)
transcription factor AP-2 alpha
E2F-like protein
+ LPS
- LPS
p65
c-Rel
RelB
p50
p52
p65
c-Rel
RelB
p50
p52
EntrezGene Description
symbol
+
+
+ +
+
+ + +w
+
+ + ++ w
+
+
+
w+
+ w
w+ +
++
w + w
w
+
+
+
+
+
+
w
+
+
+
+
+
+
+
+
+w
+
+
+
+
+
+
+
+ + +w
w+ +w
+
+ +
+
+w
+
+w
+
+
+ +
+
w
+
+
w
+ +
+
+
+
+
w
w
+
+w
+
w
++
+
w
+
+
++
+
+w
+w
+ +ww
w+ ++
+
w
+ +
+
+
+
+
+ + + +
+
X
X
X
X
w
w
+
+
w
X
w
+
+
+
w
w+ +
+ + +w
++
+ w w+ w
+
+ + ++ +
w
+
Known targets
+
+
+
+
X
X
++
+
++
+ ++
+
+
+
+
X
w
X
+
+
+ ++
TFE3
TIMP1
TJP2
TLN1
TMPIT
TNF
TNFRSF1A
TNFSF13
TNFSF13B
TNFSF14
TNIP3
TRA1
TRAF1
TRAPPC3
TRIM31
TRPM2
TTC17
UBE2H
UBE2L6
USP2
USP25
USP52
VAV1
VSIG4
WBP4
YME1L1
YWHAB
ZBTB11
ZDHHC6
ZNF217
ZNF221
ZNF581
ZNF76
ZNHIT1
transcription factor binding to IGHM enhancer 3
tissue inhibitor of metalloproteinase 1
tight junction protein 2 (zona occludens 2)
talin 1
transmembrane protein induced by tumor TNF
tumor necrosis factor (TNF superfamily, member 2)
tumor necrosis factor receptor superfamily, member 1A
tumor necrosis factor (ligand) superfamily, member 13
tumor necrosis factor (ligand) superfamily, member 13b
tumor necrosis factor (ligand) superfamily, member 14
hypothetical protein FLJ21162
tumor rejection antigen (gp96) 1
TNF receptor-associated factor 1
similar to yeast BET3 (S. cerevisiae)
tripartite motif-containing 31
transient receptor potential cation channel, subfamily M
hypothetical protein FLJ10890
ubiquitin-conjugating enzyme E2H
ubiquitin-conjugating enzyme E2L 6
ubiquitin specific protease 2
ubiquitin specific protease 25
KIAA0710 gene product
vav 1 oncogene
Ig superfamily protein
WW domain binding protein 4 (formin binding protein 21)
YME1-like 1 (S. cerevisiae)
tyrosine 3-monooxygenase, beta polypeptide
zinc finger protein
hypothetical protein FLJ21952
zinc finger protein 217
zinc finger protein 221
HSPC189 protein
zinc finger protein 76 (expressed in testis)
putative cyclin G1 interacting protein
+, Indicates high confidence binding (p< 0.002),
at least one subunit had to match this criterion
w, Indicates weaker binding events (p< 0.01)
- LPS
+
++
++
++
+
+ + +w
+ + w
+
+w
+ w
+
+w
+ + ++
++
++
+ +
+
+ w
+
+
+
+w
+
+ w++
+w
+
++
+
+
Known targets
p65
c-Rel
RelB
p50
p52
+ LPS
p65
c-Rel
RelB
p50
p52
EntrezGene Description
symbol
+
+
++
+
+
w
+
++
+
+
+
+ +
+
+
++
w
+
+
+
ww
+
+
++ +
+
+ + w
+
X
+
+
+
+
++
X
Table 4. Genes bound by E2F4 in unstimulated U937 cells
EntrezGene Description
symbol
EntrezGene Description
symbol
AK2
AP2S1
AP4B1
APG4C
ARHGAP11A
ASF1B
ATF2
ATM
ATP5G3
BAT4
BAZ1B
BCAS1
BLM
BRD8
BUB3
C10orf7
C11orf10
C12orf5
C14orf123
C20orf172
C20orf77
C21orf45
C6orf166
C6orf18
C6orf211
C6orf82
C9orf76
CASP2
CASP8AP2
CBX3
CBX5
CCNB1
CDC25A
CDC25C
CDC6
CDCA1
CDCA8
CENPF
CKAP1
CLSPN
CSTF3
DCLRE1B
DDX10
DKFZP434B168
DLG7
EIF3S2
EXO1
FANCL
FLJ10579
FLJ10922
FLJ11029
FLJ11222
FLJ13912
FLJ20354
FLJ20508
GHITM
GPHN
H2AFG
H2AFP
HAGHL
HIST1H2AJ
HIST1H2AL
HIST1H2BJ
HIST1H2BK
HIST1H2BL
HIST1H2BM
HIST1H3B
HIST1H4C
HIST1H4K
HMGB2
HNRPA1
HPCL2
HSPC052
HSPC150
HT007
IER5
IRF3
ITGA2
KIAA0042
KIAA0092
KIAA0101
KIAA0410
KIAA0747
KIAA0872
KIF20A
KIF22
KIF2C
KIF4A
KNSL7
KNTC2
KPNB1
LENG5
LGALS1
LOC55924
LOC81691
LSM4
LUC7L2
M96
MAPK7
MASTL
MCM3
MDH1
MDS009
MGC5469
MIS12
MRP63
MRPS11
MRPS15
MRPS18C
MRPS27
MTAP
MTRF1L
MUT
MYOHD1
NAP1L1
NBR2
NDUFA1
NDUFB9
NEK2
NFKBIB
NFYA
NFYC
NUDT15
NUP98
NUSAP1
ORC3L
ORMDL1
PAI-RBP1
PCNA
PCNP
PDZK11
PELO
PEX12
PIGC
PLCG2
PLK1
PLK4
PMS1
POLA
POLD1
POLE3
POLR3K
PRPS1
QPCTL
RAD51
RAMP
RBBP4
RBL1
RBM15
RFC5
RNASE4
RNPC2
RPGR
RPL26
adenylate kinase 2
adaptor-related protein complex 2, sigma 1 subunit
adaptor-related protein complex 4, beta 1 subunit
APG4 autophagy 4 homolog C (S. cerevisiae)
KIAA0013 gene product
ASF1 anti-silencing function 1 homolog B
activating transcription factor 2
ataxia telangiectasia mutated
ATP synthase, H+ transporting, mitochondrial F0 complex
HLA-B associated transcript 4
bromodomain adjacent to zinc finger domain, 1B
breast carcinoma amplified sequence 1
Bloom syndrome
bromodomain containing 8
BUB3 budding uninhibited by benzimidazoles 3 homolog
chromosome 10 open reading frame 76
chromosome 11 open reading frame 10
chromosome 12 open reading frame 5
chromosome 14 open reading frame 123
chromosome 20 open reading frame 172
chromosome 20 open reading frame 77
chromosome 21 open reading frame 45
chromosome 6 open reading frame 166
chromosome 6 open reading frame 18
chromosome 6 open reading frame 211
chromosome 6 open reading frame 82
chromosome 9 open reading frame 76
caspase 2, apoptosis-related cysteine protease
CASP8 associated protein 2
chromobox homolog 3 (HP1 gamma homolog, Drosophila)
chromobox homolog 5 (HP1 alpha homolog, Drosophila)
cyclin B1
cell division cycle 25A
cell division cycle 25C
CDC6 cell division cycle 6 homolog (S. cerevisiae)
cell division cycle associated 1
cell division cycle associated 8
centromere protein F (350/400kD, mitosin)
cytoskeleton-associated protein 1
claspin homolog (Xenopus laevis)
cleavage stimulation factor, 3' pre-RNA, subunit 3
DNA cross-link repair 1B (PSO2 homolog, S. cerevisiae)
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 10
DKFZP434B168 protein
discs, large homolog 7 (Drosophila)
eukaryotic translation initiation factor 3, subunit 2
exonuclease 1
Fanconi anemia, complementation group L
hypothetical protein FLJ10579
hypothetical protein FLJ10922
hypothetical protein FLJ11029
hypothetical protein FLJ11222
hypothetical protein FLJ13912
hypothetical protein FLJ20354
hypothetical protein FLJ20508
growth hormone inducible transmembrane protein
gephyrin
H2A histone family, member G
H2A histone family, member P
hydroxyacylglutathione hydrolase-like
histone 1, H2aj
histone 1, H2al
histone 1, H2bj
histone 1, H2bk
histone 1, H2bl
histone 1, H2bm
histone 1, H3b
histone 1, H4c
histone 1, H4k
high-mobility group box 2
heterogeneous nuclear ribonucleoprotein A1
2-hydroxyphytanoyl-CoA lyase
HSPC052 protein
HSPC150 protein similar to ubiquitin-conjugating enzyme
uncharacterized hypothalamus protein HT007
immediate early response 5
interferon regulatory factor 3
integrin, alpha 2 (CD49B)
KIAA0042 gene product
KIAA0092 gene product
KIAA0101 gene product
KIAA0410 gene product
KIAA0747 protein
KIAA0872 protein
kinesin family member 20A
kinesin family member 22
kinesin family member 2C
kinesin family member 4A
kinesin-like 7
kinetochore associated 2
karyopherin (importin) beta 1
leukocyte receptor cluster (LRC) member 5
lectin, galactoside-binding, soluble, 1
hypothetical protein
exonuclease NEF-sp
U6 snRNA-associated Sm-like protein
LUC7-like 2 (S. cerevisiae)
putative DNA binding protein
mitogen-activated protein kinase 7
microtubule associated serine/threonine kinase-like
MCM3 minichromosome maintenance deficient 3
malate dehydrogenase 1, NAD (soluble)
x 009 protein
hypothetical protein MGC5469
MIS12 homolog (yeast)
mitochondrial ribosomal protein 63
mitochondrial ribosomal protein S11
mitochondrial ribosomal protein S15
mitochondrial ribosomal protein S18C
mitochondrial ribosomal protein S27
methylthioadenosine phosphorylase
mitochondrial translational release factor 1-like
methylmalonyl Coenzyme A mutase
myosin head domain containing 1
nucleosome assembly protein 1-like 1
NBR2
NADH dehydrogenase 1 alpha subcomplex, 1
NADH dehydrogenase 1 beta subcomplex, 9
NIMA (never in mitosis gene a)-related kinase 2
NF-κB inhibitor, beta
nuclear transcription factor Y, alpha
nuclear transcription factor Y, gamma
nudix-type motif 15
nucleoporin 98kD
nucleolar and spindle associated protein 1
origin recognition complex, subunit 3-like
ORM1-like 1 (S. cerevisiae)
PAI-1 mRNA-binding protein
proliferating cell nuclear antigen
PEST-containing nuclear protein
PDZ domain containing 11
pelota homolog (Drosophila)
peroxisomal biogenesis factor 12
phosphatidylinositol glycan, class C
phospholipase C, gamma 2
Polo like Kinase 1
Polo like Kinase 4
PMS1 postmeiotic segregation increased 1
polymerase (DNA directed), alpha
polymerase (DNA directed), delta 1, catalytic subunit
polymerase (DNA directed), epsilon 3 (p17 subunit)
polymerase (RNA) III (DNA directed) polypeptide K
phosphoribosyl pyrophosphate synthetase 1
glutaminyl-peptide cyclotransferase-like
RAD51 homolog (RecA homolog, E. coli)
RA-regulated nuclear matrix-associated protein
retinoblastoma binding protein 4
retinoblastoma-like 1 (p107)
RNA binding motif protein 15
replication factor C (activator 1) 5 (36.5kD)
ribonuclease, RNase A family, 4
RNA-binding region (RNP1, RRM) containing 2
retinitis pigmentosa GTPase regulator
ribosomal protein L26
EntrezGene Description
symbol
RPL37
RPL39L
RQCD1
RRM1
RXRB
SAMHD1
SF3A3
SFRS1
SFRS2
SLC2A4
SLC31A1
SLC35A5
SLC39A9
SLC8A3
SMC1L1
SMC2L1
SMC4L1
SMN2
SNRPD2
SNX13
SPOCK3
STOML2
SUCLG1
SULT1A3
SUMO1
TCF19
TDRD3
TIMELESS
TMEM5
TOPK
TTF2
TTRAP
TU12B1-TY
TUSC4
TYMS
UCHL5
USP1
VILL
VPS29
WDR19
XRCC5
YWHAB
ZIC1
ZNF451
ribosomal protein L37
ribosomal protein L39-like
RCD1 required for cell differentiation1 homolog
ribonucleotide reductase M1 polypeptide
retinoid X receptor, beta
SAM domain and HD domain, 1
splicing factor 3a, subunit 3, 60kD
splicing factor, arginine/serine-rich 1
splicing factor, arginine/serine-rich 2
solute carrier family 2, member 4
solute carrier family 31, member 1
solute carrier family 35, member A5
solute carrier family 39, member 9
solute carrier family 8, member 3
SMC1 structural maintenance of chromosomes 1-like 1
SMC2 structural maintenance of chromosomes 2-like 1
SMC4 structural maintenance of chromosomes 4-like 1
survival of motor neuron 2, centromeric
small nuclear ribonucleoprotein D2 polypeptide (16.5kD)
sorting nexin 13
sparc, cwcv and kazal-like domains proteoglycan 3
stomatin (EPB72)-like 2
succinate-CoA ligase, GDP-forming, alpha subunit
sulfotransferase family, cytosolic, 1A, phenol-preferring 3
SMT3 suppressor of mif two 3 homolog 1
transcription factor 19 (SC1)
tudor domain containing 3
timeless homolog (Drosophila)
transmembrane protein 5
T-LAK cell-originated protein kinase
transcription termination factor, RNA polymerase II
TRAF and TNF receptor associated protein
TU12B1-TY protein
tumor suppressor candidate 4
thymidylate synthetase
ubiquitin carboxyl-terminal hydrolase L5
ubiquitin specific protease 1
villin-like
vacuolar protein sorting 29 (yeast)
WD repeat domain 19
X-ray repair complementing defective repair in Chinese hamster cells 5
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein
Zic family member 1 (odd-paired homolog, Drosophila)
zinc finger protein 451
Table 5. Genes bound by four or five NF-κB subunits
EntrezGene Description
symbol
4 subunits
APBB1IP
ASH1
BCL2A1
C3
CCL4
CCRL2
CD69
CREB1
DBI
EMP3
FLJ10420
FLJ22555
HSPCB
IL1F9
MAN2B1
OAS3
P4HA1
PPP1R15B
TAPBP
TIMP1
TNF
WBP4
similar to proline-rich protein 48
hypothetical protein ASH1
BCL2-related protein A1
complement component 3
small inducible cytokine A4
chemokine (C-C motif) receptor-like 2
CD69 antigen (p60, early T-cell activation antigen)
cAMP responsive element binding protein 1
diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein)
epithelial membrane protein 3
hypothetical protein FLJ10420
hypothetical protein FLJ22555
heat shock 90kD protein 1, beta
interleukin-1 homolog 1
mannosidase, alpha, class 2B, member 1
2'-5'-oligoadenylate synthetase 3 (100 kD)
procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I
protein phosphatase 1, regulatory (inhibitor) subunit 15B
TAP binding protein (tapasin)
tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor)
tumor necrosis factor (TNF superfamily, member 2)
WW domain binding protein 4 (formin binding protein 21)
5 subunits
AQP9
ARHGEF2
CCL3
CCNL1
DAF
FLJ20477
FLOT1
GADD45B
GPX1
GYPC
ICAM1
IL2RG
NFKB2
SMPD1
TRAF1
aquaporin 9
rho/rac guanine nucleotide exchange factor (GEF) 2
small inducible cytokine A3, Mip-1-alpha
cyclin L ania-6a
decay accelerating factor for complement (CD55)
hypothetical protein FLJ20477
flotillin 1
growth arrest and DNA-damage-inducible, beta
glutathione peroxidase 1
glycophorin C (Gerbich blood group)
intercellular adhesion molecule 1 (CD54)
interleukin 2 receptor, gamma
p52/p100
sphingomyelin phosphodiesterase 1, acid lysosomal
TNF receptor-associated factor 1
Supporting Figure Legends
Figure 6. Example scatter plots showing binding of p65 and p50 to gene
promoters in living cells.
Microarray data from p65 ChIP in unstimulated cells and p50 ChIP following LPS
stimulation. The x-axis represents the hybridization signal intensity for input
whole cell extract (WCE) DNA (log10 Cy3 background-subtracted signal). The yaxis shows the intensity for DNA enriched by ChIP using antibodies specific for
p65 or p50 (log10 Cy5 background-subtracted signal). Microarray elements that
were flagged as not found by the Axon software were not analyzed. The red lines
either side of the scatter plots represent the ratio of IP signal to WCE signal
necessary to achieve a significance score of 0.002. The position of the promoters
for the known NF-κB target genes HLA-B, NFKB2, CCL1, PTX3, CSF2 and
RAC2, the novel NF-κB target genes NFKBIB and P4HA1 and β-actin, a negative
control, are marked in both scatter plots. No genes were enriched above the pvalue threshold of 0.002 by p65 ChIP in unstimulated cells. In contrast, HLA-B,
NFKB2, CCL1, PTX3, CSF2, RAC2, NFKBIB and P4HA1 were among the 193
genes enriched above this threshold by p50 ChIP in stimulated cells. β-actin was
not enriched in either p65 or p50 ChIP.
Figure 7. Gene-specific PCRs of immunoprecipitated and whole cell extract
DNA.
A) Gene-specific PCRs of promoters at which binding was detected using the
promoter arrays. All gene-specific PCRs were performed with DNA from LPS-
stimulated cells. 1/30th of the immunoprecipitated (IP) DNA (equivalent to 1.5x106
cells) was amplified with the designated promoter specific primers and the results
compared to the amplification of serial dilutions of whole cell extract DNA
(1/18000th, 1/6000th and 1/2000th). Thus, a similar intensity DNA band in the IP
amplification and the highest WCE dilution amplification indicates that the DNA
sequence is present in the IP fraction at an amount ~600x lower than in the
WCE. We amplified the β-actin promoter from the IP and WCE DNA as a
negative control (β-actin was not bound by NF-κB in the array results, see
Supporting Information Figure 6). The intensities of the bands were quantified
using ImageQuant (Amersham). The enrichment ratio is the average foldenrichment of the promoter of interest in the IP fraction compared to WCE,
normalized to the β-actin control (ie. a ratio greater than 1 indicates that the ratio
of IP to WCE is greater for the NF-κB target gene than for β-actin).
B) Gene-specific PCRs of promoters at which binding was not detected using the
promoter arrays.
C) Intersection between previously known directly bound NF-κB target genes and
targets identified in this study. The complete list of previously known targets is
compiled from all the cell types and conditions in the literature and is available
through the Literature Targets link at web.wi.mit.edu/young/nfkb (based on Pahl,
H.L. (1999). Oncogene 18, 6853-6866 and the Gilmore lab database at
http://people.bu.edu/gilmore/nf-kb/target/index.html). Of the 224 literature targets,
our array covered the promoters of 154 genes. Of these, 38 NF-κB targets were
bound at a p-value of 0.002 (24%, p=4x10-19) and 49 at a p-value of 0.01 (32%,
p=2x10-19). As a negative control, we compared the NF-κB literature targets to
those identified for E2F4 using location analysis and found no significant overlap
(p=0.96). A list of the E2F4 bound genes can be found in Supporting Information,
Table 4.
Figure 8. Immunoblotting of NF-κB proteins in the cytoplasm and nucleus
of U937 cells before and after LPS stimulation.
U937 cells were treated with LPS (2.5 µg/ml) and nuclear and cytosolic extracts
were harvested at 0, 30, 60 and 180 minutes following stimulation. p50 and p52
were both present in the nuclei of unstimulated cells and increased in nuclei after
LPS stimulation, consistent with the results of location analysis. The p52 antibody
(Upstate, 06-413) detects an additional band in the nucleus corresponding to a
protein of approximately 58 kD. This protein is unlikely to be an NF-κB subunit
because it is not present in the cytoplasm and does not increase upon LPS
stimulation. This unspecific band is also apparent in western blots performed
using this antibody by other investigators (Senftleben et al. 2001. Science
293,1495-1499). We do not believe that this unspecific binding significantly
affects our location analysis results because the vast majority of the p52 targets
we identify were also detected by antibodies to other NF-κB subunits, either
before or after LPS stimulation. Small amounts of RelB and c-Rel are apparent in
the nuclei of unstimulated U937 cells after long exposures to film. We also detect
a relatively small number of targets for these proteins in unstimulated cells
(Figure 1). Location analysis can be more sensitive than western blotting for
small amounts of DNA binding proteins because the technique uses
immunoprecipitation to concentrate protein from ~5x107 cells and then LM-PCR
to amplify co-precipitating DNA.
Materials and methods: Extracts were prepared using a protocol modified from
Dignam and colleagues (Dignam et al. (1983). Nucleic Acids Res. 11, 14751489). Cells were washed with PBS and lysed on ice in hypertonic buffer with
NP-40 (10mM Hepes, pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 0.1% NP40 with Complete protease inhibitor (Roche)) for 5 minutes. The cytosolic fraction
was harvested by centrifuging the lysate at 4500g for 3 min and collecting the
supernatant. The nuclear pellet was washed twice in hypertonic buffer without
NP-40. Nuclear proteins were then extracted on ice in high-salt buffer (20mM
Hepes, pH 7.9, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM DTT, 10%
glycerol with Complete protease inhibitor (Roche)) for 30 minutes. The nuclear
extract was harvested by centrifuging the lysate at 7000g for 3 min and collecting
the supernatant. Protein concentrations were determined using the BCA kit
(Pierce) and 8µg of protein extracts were loaded per lane and separated by SDSpolyacrylamide gel electrophoresis. Proteins were transferred onto Hybond-P
membrane (Amersham) and the membrane blocked with 5% milk in PBS
containing 0.1% Tween-20 (Sigma). The primary antibodies used were identical
to those used for location analysis. The blots were developed with ECL
(Amersham).
Figure 9. Recruitment of activatory NF-κB subunits to activated genes upon
LPS stimulation.
A. NF-κB binding at genes to which RNA polymerase II is recruited and to genes
where it is lost (top 5% of genes for both categories). The change in RNA
polymerase binding is represented as a ratio, with red indicating an increase and
green a decrease, according to the scale on the left. Only genes bound by RNA
polymerase II in either unstimulated or activated cells were used. NF-κB subunits
that were already bound (p<0.002) before LPS stimulation are marked in blue
and the recruitment of new subunits is marked in red.
B. NF-κB binding at genes that are upregulated or downregulated over time in
response to LPS in U937 cells. Genes were either upregulated by 2-fold or
downregulated by 2-fold at two consecutive time points. Red indicates an
increase in expression and green a decrease according to the scale on the left.
Figure 10. Model of the transcriptional regulatory network coordinated by
NF-κB family members.
Genes (boxes) bound by NF-κB subunits (circles) in U937 cells before and after
stimulation with LPS. Among the NF-κB target genes identified here are many
transcription factors with roles in regulating the immune response. Therefore we
suggest a model in which NF-κB controls the response to pathogens, not only by
binding to its direct targets, but also through the activation of other transcription
factors.
Supporting Information Figure 6
log(IP)
log(IP)
0 targets
4
p65
-LPS
HLA-B
NFKB2
NFKBIB
CCL1
P4HA1
PTX3
β-actin
CSF2
RAC2
3
2
3
2
1
0
193 targets
4
p50
+LPS
HLA-B
NFKB2
NFKBIB
P4HA1
CCL1
CSF2
RAC2
PTX3
β-actin
1
0
1
2
3
44
log(WCE)
0
0
1
2
3
4
log(WCE)
Supporting Information Figure 8
Cytoplasm
0 30 60 180
p65
p105
p50
RelB
c-Rel
p100
p52
Nucleus
0 30 60 180
NF-κB
binding
B
2
5/3
3/2
2/3
3/5
1/2
>8
4
2
1
1/2
1/4
<1/8
Change in gene
expression
NF-κB
binding
p50
p52
p65
RelB
c-Rel
Change in
RNA Pol II
p50
p52
p65
RelB
c-Rel
A
0 hrs
1 hr
3 hrs
9 hrs
27 hrs
Supporting Information Figure 9
Supporting Information Figure 10
RB1
BCL6
p50
PRDM1 TAPBP
IL8
JUN
p52
STAT1
NFKBIB
TFAP2A
p65
VAV1
RB1
ICAM1 STAT6 CDKNA1
RelB
CREB1 CCL3 CCL4
+ LPS
c-Rel
SRF
NFKB2
BCL6
p50
PRDM1 TAPBP
IL8
JUN
p52
STAT1
NFKBIB
TFAP2A
p65
VAV1
ICAM1 STAT6 CDKNA1
RelB
CREB1 CCL3 CCL4
c-Rel
SRF
NFKB2