www.sciencemag.org/cgi/content/full/317/5838/675/DC1

Supporting Online Material for

Negative Regulation of Toll-Like Receptor Signaling by NF-κB p50 Ubiquitination Blockade

Ruaidhrí J. Carmody, Qingguo Ruan, Scott Palmer, Brendan Hilliard, Youhai H. Chen*

*To whom correspondence should be addressed. E-mail: yhc@mail.med.upenn.edu

Published 3 August 2007, Science 317, 675 (2007) DOI: 10.1126/science.1142953

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S9 References

1. Materials and Methods Animals. The Bcl3-/- B6,129 mice (1, 2) were initially obtained from the Jackson Laboratory. They were backcrossed to C57BL/6J mice for 12 generations before being used in this study. Age- and sex-matched wild type littermates were used as controls. All animal procedures were pre-approved by the University of Pennsylvania Animal Care and Use Committee.

Bcl3-/- mice have severe defects in the microarchitecture of their secondary lymphoid organs including reduced germinal centers and follicular dendritic cell networks (1, 2), which in turn affect the immune competence of these animals. The structural defect is not present in chimeric mice reconstituted with Bcl3-/- bone marrow (3). In this study, bone marrow chimeric mice were generated by irradiating C57BL/6 mice twice with 500 rads spaced three hours apart, followed by i.v. injection of 107 bone marrow cells from wild type or Bcl3-/- C57BL/6 mice. Repopulation of the immune system was monitored by flow cytometric analysis of the blood. As we reported, in the chimeric mice so generated, ~90% of the T cells and >95% of the B cells and myeloid cells were derived from donor bone marrow 8-9 weeks after the cell transfer (4). Use of these bone marrow chimeric mice excludes the possibility that the structural defect in Bcl3-/- mice directly affects LPS tolerance and sepsis. Flow cytometric analysis.

Cells were washed in PBS, incubated for 15 min on ice with antibodies to CD40, CD80, CD86, CD11b and MHC II I-Ab (BD Biosciences), and analyzed on a BD FACScalibur. Phagocytosis assay.

Cells were incubated with fluorescent latex Fluorospheres (Molecular Probes) at a dilution of 0.05% for 2 hrs, washed and analyzed on a BD FACScalibur. Cell culture, plasmids and transfection.

Resident and elicited peritoneal macrophages were obtained by peritoneal lavage of untreated mice and mice injected intraperitoneally three days earlier with 1ml of 4% thioglycollate broth, respectively. Following isolation, peritoneal macrophages were cultured in DMEM containing 10% FBS, 2mM glutamine, and 100U/ml penicillin/streptomycin with or without 100 ng/ml LPS. B220+ B cells were isolated from spleen of naïve mice using the AutoMacs magnetic cell sorter (Miltenyi Biotec, Auburn, CA) following incubation of splenocytes with anti-mouse B220 mAb. Bone marrow-derived dendritic cells were prepared as we described (4). Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM containing 10% FBS, 2mM glutamine, and 100U/ml penicillin/streptomycin. Transfections were performed using Fugene-6 according to the manufacturer’s instructions (Roche). Mammalian expression vectors for Bcl-3 and p50 were generated following PCR amplification and ligation of murine cDNAs into pcDNA3.1Myc and pEF4-Xpress vectors. The DNA binding-defective mutant p50Y57AG60D was generated using the Quick-change kit according to the manufacturer’s instructions (Stratagene). HA-ubiquitin and the K48R, K63R and Kθ

1

mutants were generous gifts from M. Walsh (University of Pennsylvania, Philadelphia, PA). Western blot and ubiquitination assay. For Western blot, whole cell lysates were extracted from cells suspended in RIPA buffer (50mM Tris-HCl pH7.4, 1% NP-40, 0.25% deoxycholate, 150mM NaCl, 1mM EDTA, 1mM sodium orthovanadate) supplemented with 1X Complete protease inhibitor cocktail (Roche). Nuclear extracts were obtained using the Nuclear Extract kit according to the manufacturer’s instructions (Active Motif). Lysates were resolved on SDS-PAGE gels, transferred to nitrocellulose membranes, and blotted with specific antibodies. The following antibodies were purchased from Cell Signaling Technology: anti-phospho IκBα(Ser32), anti-phospho-ERK1/2(Thr202/tyr204), anti-ERK1/2, anti-phospho-JNK(Thr183/Tyr185), anti-JNK, anti-phospho-p38(Thr180/Tyr182) and anti-p38. Anti-IκBα was purchased from Upstate (NY), anti-p50 from Stressgen Bioreagents (Victoria, Canada), anti-p65(C-20) and anti-c-Rel(C) were from Santa Cruz Biotech. For Ubiquitin assays, HEK293T cells were transiently transfected with xpress-p50, HA-Ubiquitin or indicated mutants, and myc-Bcl-3 for 24 hours. Cells were then incubated with 10µM N-ethylmaleamide (N-EM) for 30 seconds, washed in PBS/10µM N-EM, lyzed in 1% SDS supplemented with 1X complete protease inhibitor cocktail (Roche), boiled for 5 min, and diluted (1/10) in RIPA buffer supplemented with 20µM N-EM and 1X protease inhibitors. Equal amounts of protein were precleared with protein-G agarose beads, immunoprecipitated with an anti-xpress antibody (Invitrogen) and analyzed by Western blot using an anti-HA Ab (Roche) to detect ubiquitinated p50 species. To detect ubiquitination of endogenous p50 in bone marrow-derived macrophages, cells were pretreated with MG132 (20µM) for 30mins at 37°C and washed in PBS/10µM N-EM before lysis. Equal amounts of protein were precleared with protein-G agarose beads before immunoprecipitation with anti-p50 antibody (Biostressgen). Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane and autoclaved for 40 mins prior to immunoblotting with an anti-ubiquitin antibody (Chemicon). 35S-Methionine/cysteine pulse chase labeling. Cells were washed twice in warm PBS and incubated in methionine/cysteine-free DMEM containing 5% dialyzed FBS and 2mM glutamine for 30 mins. Cells were then pulsed with approximately 500µCi 35S-methionine/cysteine for 60 mins, washed with warm HBSS containing 2mM methionine and 2mM cysteine and further incubated with DMEM containing 10% FBS, 2mM methionine and 2mM cysteine for the indicated times. Upon harvesting, cells were washed in ice-cold PBS, pelleted and stored at -80°C until immunoprecipitation was performed as described above. Immunoprecipitates were resolved by SDS-PAGE, dried and exposed to a phosphor screen overnight prior to analysis on a Phosphoimager. Gene expression analysis. For real-time PCR, total RNA was isolated using RNeasy kits (Qiagen) primed with random hexamer oligonucleotides and reversely transcribed using Invitrogen First Strand cDNA synthesis kit. PCR was performed using FAM-labeled probe sets for

2

cytokines (Applied Biosciences), a universal master mix and the ABI7900 or SYBR Green master mix and the following primer sets: SOCS1, forward 5-CAG GTG GCA GCC GAC AAT GCG ATC-3, and reverse 5-CGT AGTGCT CCA GCA GCT CGA AAA-3; IRAK-M, forward 5′-TGAGCAACGGGACGCTTT-3′, and reverse 5′-GATTCGAACGTGCCAGGAA-3′; ST2, forward 5-ACGCTCGACTTATCCTGTGG-3, and reverse 5-CAGGTCAATTGTTGGACACG-3; A20, forward 5’-GGAGA CGGGACTTTGCTACGA-3’, and reverse 5’-GTGTGTCTGCTGAGGCCATTT-3’. All data were normalized to 18s rRNA. For protein microarray, culture supernatants were collected and tested using the Raybiotech cytokine antibody microarray (Raybiotech, Norcross, GA). The strengths of the signals were determined by densitometry. Chromatin immunoprecipitation (IP). Chromatin immunoprecipitation was performed using the ChIP assay kit from Upstate Biotech according to the manufacturer’s instructions. Antibodies used include anti-p50 (AbCam), anti-p65 (C-20) and anti-c-Rel (C) from Santa Cruz Biotech. Primers employed are as follows: TNFα forward, 5’-GAGGCTCCGTGGAAAACTCACTTG-3’; TNFα reverse, 5’-GCAGAGCAGCTTGAGAGTTGGGAA-3’; CXCL2 forward, 5’-ACATCCCAGGGTCCCATAGTGGAA-3’; CXCL2 reverse, 5’-AGGAAGCTTGTT GGAGGCACTGA-3’. Electrophoretic mobility shift assay (EMSA). NF-κB consensus double stranded oligonucleotides (5’-AGTTGAGGGGACTTTCCCAGG-3’) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). They were first end-labeled with [γ32P]ATP (Amersham Biosciences, Piscataway, New Jersey) using the T4 polynucleotide kinase (Promega, Madison, Wisconsin). Binding reactions were prepared using 1 to 5µg of nuclear extract with 50,000 cpm of oligonucleotides in a 25µl reaction volume containing 10mM HEPES-KOH (pH7.9), 50mM KCL, 2.5mM MgCl2, 1mM DTT, 10% glycerol, 1µg DNAse free bovine serum albumin and 2.5µg poly[d(I-C)] at room temperature for 30 mins. For supershift analysis, antibodies against p50, p65(C-20) and c-Rel(C) (Santa Cruz Biotech) were added to the reaction mixture on ice for 20 min prior to the addition of radiolabeled probes. Binding reactions were resolved on a 4% non-denaturing polyacrylamide gel at 22mA for 3 hours at 4°C in 1x TBE (0.089 Tris-borate, 0.089M boric acid and 0.002M EDTA). Gels were subsequently dried and exposed to a phosphor screen and visualized on a phosphoimager (Amersham Biosciences). Densitometry was performed using ImageQuant 5.2 software (Amersham Biosciences). Statistical analysis. The differences in proteins, mRNA, and promoter reporter activities were analyzed by Student’s t test. The differences in survival rates were analyzed by Mann-Whitney U test. For experiments involving mice, a minimum of 3 mice per group were used.

3

2. Supporting Text NF-κB is a master regulator of inflammatory gene expression and the core

component of the TLR-mediated signaling pathways. Individual TLR pathways converge at the IKK complex through various combinations of adaptor molecules (5). Regulation of NF-κB is central to the regulation of TLR responses (6). At the chromatin level, gene expression is regulated by the dynamic interaction between various NF-κB subunits and their target promoters during the course of the TLR response (7, 8). NF-κB dimer exchange is believed to shape the transcriptional output from target genes by means of the intrinsic transactivation potential of particular NF-κB dimers. Limiting NF-κB transcriptional activity following TLR ligation is achieved in part by IKKα kinase activity, which triggers NF-κB ubiquitination and degradation in the nucleus (9-11), and through the IκB family of proteins which direct NF-κB nuclear export (12, 13). NF-κB-dependent expression and de novo synthesis of IκB proteins following their degradation in the cytoplasm is a key autoregulatory feedback mechanism for controlling NF-κB activity (14).

The NF-κB family of transcription factors is comprised of five members, c-Rel, RelA (p65), RelB, p50 and p52, which are capable of hetero- and homo-dimerization. Both p50 and p52 are formed by limited processing of their precursors (p105 and p100, respectively). They do not contain the transactivation domain found in other NF-κB subunits (15). Bcl-3 is an atypical member of the IκB family of proteins which resides predominantly in the nucleus and is not degraded upon NF-κB activation (16, 17). Bcl-3 interacts only with NF-κB p50 and p52 homodimers (18, 19) and its role in the regulation of NF-κB function to date has been unclear. Bcl-3 has been reported to both enhance and impede p50 homodimer DNA binding and has been found to both negatively and positively regulate NF-κB mediated gene expression (17, 20-26). Because p52 does not play a role in TLR signaling and appears to be restricted to the alternative pathway of NF-κB activation, p50 homodimer is likely the major target of Bcl-3 action in TLR signaling. However, whether Bcl-3 regulates other aspects of immunity through p52 is not clear.

In this paper, we report that Bcl-3 is a critical regulator of TLR-induced gene expression in macrophages. Bcl3-/- cells are hyper-responsive to TLR activation and express increased levels of cytokines and chemokines following TLR stimulation. Bcl3-/- macrophages display reduced p50 homodimer DNA binding due to increased p50 homodimer ubiquitination and degradation. In vitro analysis established Bcl-3 as a positive regulator of p50 homodimer DNA binding through the stabilization of DNA bound complexes. These data reveal a critical regulatory function for transcriptionally inactive p50 homodimers whose activation occurs concomitantly with p65- and c-Rel-containing dimers following TLR activation. However, although p50 homodimers are primed to limit the expression of NF-κB inducible genes in response to LPS, they are ineffective in the absence of Bcl-3.

It is to be noted that significant ubiquitination of endogenous p50 was detected only after LPS stimulation. This is because the vast majority of endogenous p50 are maintained in the cytoplasm through association with IκB proteins and are therefore unable to bind to DNA. Because DNA binding is required for triggering its ubiquitination

4

(Figure 3), cytoplasmic p50 does not undergo ubiquitination. LPS stimulation induces the degradation of IκB proteins, allowing the translocation of p50 into the nucleus where it binds to DNA and undergoes ubiquitination. By contrast, when overexpressed, p50 constitutively translocates into nucleus because there are not enough IκB proteins in the cytoplasm to retain them. Therefore, LPS is not required for inducing DNA binding and subsequent ubiquitination of overexpressed p50. Because ubiquitination of p50 plays a crucial role in regulating its function, the nature of the ubiqutin ligase involved needs now to be examined. Similarly, the state of LPS tolerance in bone marrow chimeric mice that express lysine-less p50, or DNA binding-deficient p50, in the absence of wild type p50, needs to be studied.

Recent studies have established a highly dynamic model for protein-chromatin interactions involving the NF-κB members p65 and c-Rel (27). These interactions are transient and allow promoters to sample the nucleoplasmic binding activity. A p65 mutant defective in signal-induced proteasomal degradation was found to reside on promoters for a long period of time, strongly supporting a role for proteasome-mediated turnover in the removal of NF-κB dimers from promoters (8, 10, 27). The ubiquitin-mediated degradation of DNA bound p50 homodimers described here provides further evidence for a critical role of protein turnover in the regulation of NF-κB-mediated gene expression. The rapid turnover and cycling may allow for the clearance of promoters between successive rounds of activation, and offers potential regulatory checkpoints to rapidly repress or fine-tune transcription. The inhibition of p50 homodimer ubiquitination by Bcl-3 establishes a stable DNA bound complex, which regulates the equilibrium of NF-κB interaction on promoters. Thus, promoter occupancy by stable p50 homodimer:Bcl-3 complexes is sufficient to limit gene expression controlled by transactivation-competent NF-κB complexes. The effect of Bcl-3 deficiency on the order of NF-κB dimer binding on the TNFα and CXCL2 promoters demonstrates that the stability of DNA bound complexes is a critical factor in determining the kinetics of dimer exchange. The shorter occupancy time of the p50 homodimer on NF-κB sites in the absence of Bcl-3 leads to an increased promoter occupancy by p65 and c-Rel, and increased transcriptional output. Precisely why the stabilized p50 homodimer:Bcl-3 complex is critical for correct dimer exchange is unclear. It is possible that p50 homodimer occupancy of NF-κB sites generates a pause in transcriptional complex assembly and allows NF-κB dimers to interact with or be modified by soluble nucleoplasmic factors not associated with the DNA bound complexes. Such a pause may allow for the modification of the NF-κB subunits, fine-tuning transcriptional output or facilitating interactions with IκB proteins.

The function of Bcl-3 extends beyond the inhibition of proinflammatory gene expression following acute LPS exposure. Bcl3-/- mice and macrophages are defective in LPS tolerance following chronic and repeated exposure to LPS. Bcl-3 promotes a persistent presence of inhibitory p50 homodimers on tolerized promoters after repeated exposure to LPS. These stabilized complexes could generate a state of promoter tolerance because p50 homodimer overexpression led to a long-lasting repression of TNF-α gene (28). Although Bcl-3 has been found to be associated with nuclear corepressor complexes, it has also been identified in coactivator complexes, indicating that corepressor complex formation is not dependent on Bcl-3. Indeed, the formation of

5

corepressor complexes at NF-κB responsive promoters does not require p50 homodimers and has also been described in association with p65/p50 heterodimers (29). This suggests that Bcl-3 does not play an essential role in the corepressor complexes.

In conclusion, Bcl-3 is a critical regulator of NF-κB-mediated gene expression following TLR stimulation. Bcl-3 enhances the formation of DNA bound complexes by inhibiting p50 ubiquitination and degradation. In Bcl3-/- cells, loss of stable p50 homodimers results in disrupted promoter loading and dimer exchange on NF-κB regulated genes, leading to increased inflammatory gene expression. After chronic exposure to LPS, Bcl-3 enforces p50 homodimer DNA binding on the TNF-α promoter to prevent further transactivation, thereby revealing itself as an essential regulator of LPS tolerance. Thus, Bcl-3-stabilized p50 homodimer DNA binding plays a critical role in the normal functioning of the immune system.

6

3. Supporting Figures.

Figure S1. Normal cell surface marker expression and phagocytosis of Bcl3-/- macrophages. A. Peritoneal (PM) and bone marrow-derived (BM) macrophages were isolated from wild type (solid thick line) and Bcl3-/- (dashed line) mice and stained with indicated antibodies. Cells stained with isotype-matched control antibodies are shown in thin line. B. Wild type and Bcl3-/- peritoneal macrophages were incubated with (filled histogram) or without (open histogram) fluorescent micro-beads for 2 hours, washed extensively and analyzed by flow cytometry.

7

Figure S2. Bcl-3 deficiency increases cytokine protein expression both in vivo and in vitro. A. In vivo cytokine production. Six-to-eight week old wild type and Bcl-3-/- mice (n=3), were injected with 10mg/kg LPS intraperitoneally and their sera collected 1.5 and 3 hrs later. Serum IL-6 and TNF-α levels were determined by ELISA. Data presented are means ± SEM. The differences between the two groups are statistically significant (p<0.01) for all time points except for IL-6 at 1.5 hrs. B. In vitro cytokine production. Peritoneal macrophages from wild type and Bcl3-/- mice (n=3) were stimulated with LPS for 24 hrs. Supernatants were analyzed using a cytokine protein array (Ray Biotech) according to the manufacturer’s instructions. Densitometry was used to measure the relative levels of the proteins expressed. Data presented are the means ± SEM. The differences between the two groups are statistically significant (p<0.01) for all cytokines.

8

Figure S3. Macrophage gene expression induced by TLR ligands, IL-1β and TNF-α. WT and Bcl3-/- BMD macrophages were stimulated with A) CpG (1µM), B) peptidoglycan (10µg/ml), C) poly(I:C) (50µg/ml), D) TNF-α (20ng/ml), E) IL-1β (2ng/ml), and F) LPS (100ng/ml) for the indicated times. For panel F, the NF-κB inhibitor (I) BAY11-7085 (20µM) was included in two groups (open and filled squares). Gene expression levels were determined by real-time PCR. Data shown are means±SD of triplicate samples and are representative of three independent experiments.

9

Figure S4. Normal upstream signaling and NF-κB nuclear translocation following LPS stimulation of Bcl3-/- macrophages. A. Macrophages from wild type and Bcl3-/- mice (n=3) were stimulated with 100ng/ml LPS for the indicated times prior to lysis and immuno-blotting with specific antibodies to the indicated proteins. B. Wild type and Bcl3-/- macrophages were stimulated with 100ng/ml LPS for the indicated times. Their nuclear extracts were prepared and analyzed for p50, p65 and c-Rel proteins by Western blot.

10

Figure S5. The p50Y57AG60D mutant interacts with Bcl-3 and p65, but not DNA. A. 293T cells were transfected with expression plasmids for p50Y57AG60D, Bcl-3 and p65. p50Y57AG60D was immunoprecipitated (IP) from the cell lysates and analyzed by Western blot (WB) for Bcl-3 and p65. Lysates were also analyzed by WB to demonstrate equal expression of the proteins. B. 293T cells were transfected with expression plasmids as indicated. Nuclear extracts were prepared from the transfected cells and tested in an EMSA using the consensus NF-κB binding nucleotides. WB of nuclear lysates confirmed the expression of all proteins.

11

Figure S6. Reduced p50 half-life, normal TLR proximal signaling and normal expression of TLR regulators in tolerized Bcl3-/- macrophages. A. Tolerized wild type and Bcl3-/- BMD macrophages were pulse-labeled with 35S-methionine/cysteine and restimulated with LPS (100ng/ml). p50 protein was chased for the indicated times and its half-life (t1/2) calculated from a one-phase exponential decay plot of band intensity. B. Tolerized wild type and Bcl3-/- BMD macrophages were re-stimulated with LPS (100ng/ml) for the indicated times. Cell lysates were immunoblotted with antibodies to the indicated proteins. C. Wild type BMD macrophages were either left untreated (Unt) or tolerized (Tol) with LPS. Bcl-3 mRNA was quantified by real-time PCR. D. Wild type and Bcl3-/- BMD macrophages were either left untreated or tolerized with LPS. Selected mRNAs were quantified by real-time PCR. Data shown are means and SD of triplicate samples.

12

Figure S7. Bcl-3 knockdown prevents LPS tolerance. A. Myc-Bcl-3-expressing 293T cells were transfected with the pSilencer hygro vector (Ambion) containing negative control siRNA or siRNA against the murine Bcl-3 target sequence 5’-ATAACATAGCCGCTGCTA-3’ according to the manufacturer’s instructions. The ability of siRNA to knockdown Bcl-3 expression was assessed by immunoblotting against myc-tagged Bcl-3. Immunoblotting against the related IκBα protein was used as a loading control. B. Luciferase assay using TNF-α promoter reporter plasmid in RAW cells transfected with control or Bcl-3 siRNA. Raw cells were pretreated with (+) or without (-) 100ng/ml LPS for 24 hours, washed and restimulated with the same concentration of LPS for an additional 8 hrs. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was used as an internal control. Reporter activity is presented as fold increase over unstimulated cells. LPS pretreatment reduced the reporter activity by 80% in the control group and by 40% in the Bcl-3 siRNA group. The difference between the two groups is statistically significant (p<0.004). Data presented are mean + SEM of triplicate cultures and are representative of three independent experiments.

13

Figure S8. Bcl-3 inhibits TNF-α promoter activity in RAW macrophages. RAW cells were co-transfected with empty expression vector or Bcl-3 expression vector along with TNF-α luciferase reporter plasmid. Renilla luciferase expression vector was included to normalize the transfection efficiency across samples. Twenty-four hours post transfection, cells were cultured with (+) or without (-) LPS for 8 hrs and luciferase activity analyzed. Reporter activity is presented as fold increase over unstimulated cells. Data presented are mean + SEM of triplicate cultures and are representative of three independent experiments. The difference between the two groups is statistically significant for LPS-treated cells (p<0.0001).

14

A. B.

FthcmblepcaTPre

p50

p50

QuickTime™ and a decompressor

are needed to see this picture.

Bcl-3

p50p50

mRNA

c-Rel p65

p50p50

Ubiquitination

Degradation

c-Rel p65

c-Rel p65

DNAmRNA

p50

p50

QuickTime™ and a decompressor

are needed to see this picture.

Bcl-3

igure S9. Schematic view of the dual suppressor model proposed in this paper. A. In e absence of Bcl-3, a gene promoter is occupied primarily by the stimulatory NF-κB

omplex, c-Rel:p65, following TLR activation; the inhibitory complex, p50:p50 dimer, ay also bind to the promoter but the p50:p50:DNA complex is inherently unstable

ecause p50 binding to DNA quickly target it to ubiquitination and degradation. This ads to sustained activation of the gene promoter. B. In the presence of Bcl-3, the 50:p50:DNA complex is stabilized by the formation of the Bcl-3:p50:p50:DNA omplex, which is resistant to ubiquitination. This reduces the number of κB sites vailable to stimulatory NF-κB dimers, limiting the degree of the promoter activation. hus, in this model, Bcl-3 serves as a stabilizer of the inhibitory p50:p50:DNA complex. romoter tolerance is generated by extending the life of the inhibitory complex and quires the presence of both Bcl-3 and p50 (dual factors).

15

4. Supporting References 1. E. M. Schwarz, P. Krimpenfort, A. Berns, I. M. Verma, Genes Dev 11, 187

(1997). 2. G. Franzoso et al., Immunity 6, 479 (1997). 3. L. Poljak, L. Carlson, K. Cunningham, M. H. Kosco-Vilbois, U. Siebenlist, J

Immunol 163, 6581 (1999). 4. R. J. Carmody, B. Hilliard, K. Maguschak, L. A. Chodosh, Y. H. Chen, J

Neuroimmunol 133, 95 (2002). 5. S. Akira, K. Takeda, Nat Rev Immunol 4, 499 (2004). 6. S. Gerondakis, M. Grossmann, Y. Nakamura, T. Pohl, R. Grumont, Oncogene 18,

6888 (1999). 7. S. Saccani, S. Pantano, G. Natoli, J Exp Med 193, 1351 (2001). 8. S. Saccani, S. Pantano, G. Natoli, Mol Cell 11, 1563 (2003). 9. T. Lawrence, M. Bebien, G. Y. Liu, V. Nizet, M. Karin, Nature 434, 1138 (2005). 10. S. Saccani, I. Marazzi, A. A. Beg, G. Natoli, J Exp Med 200, 107 (2004). 11. A. Ryo et al., Mol Cell 12, 1413 (2003). 12. F. Carlotti, S. K. Dower, E. E. Qwarnstrom, J Biol Chem 275, 41028 (2000). 13. W. F. Tam, L. H. Lee, L. Davis, R. Sen, Mol Cell Biol 20, 2269 (2000). 14. S. Ghosh, M. Karin, Cell 109, S81 (2002). 15. S. Beinke, S. C. Ley, Biochem J 382, 393 (2004). 16. L. D. Kerr et al., Genes Dev 6, 2352 (1992). 17. F. G. Wulczyn, M. Naumann, C. Scheidereit, Nature 358, 597 (1992). 18. G. P. Nolan et al., Mol Cell Biol 13, 3557 (1993). 19. J. Inoue, T. Takahara, T. Akizawa, O. Hino, Oncogene 8, 2067 (1993). 20. G. Franzoso et al., Nature 359, 339 (1992). 21. V. Bours et al., Cell 72, 729 (1993). 22. G. Franzoso et al., Embo J 12, 3893 (1993). 23. T. Fujita, G. P. Nolan, H. C. Liou, M. L. Scott, D. Baltimore, Genes Dev 7, 1354

(1993). 24. S. Grundstrom, P. Anderson, P. Scheipers, A. Sundstedt, J Biol Chem 279, 8460

(2004). 25. R. A. Corn, C. Hunter, H. C. Liou, U. Siebenlist, M. R. Boothby, J Immunol 175,

2102 (2005). 26. M. Riemann, R. Endres, S. Liptay, K. Pfeffer, R. M. Schmid, J Immunol 175,

3560 (2005). 27. D. Bosisio et al., Embo J 25, 798 (2006). 28. J. Bohuslav et al., J Clin Invest 102, 1645 (1998). 29. S. H. Baek et al., Cell 110, 55 (2002).

16