Pellino 1 Is Required for Interleukin-1 (IL-1)-mediated
Signaling through Its Interaction with the IL-1 Receptor-associated
Kinase 4 (IRAK4)-IRAK-Tumor Necrosis Factor Receptor-associated Factor
6 (TRAF6) Complex*
Zhengfan
Jiang
,
H. Jan
Johnson§,
Huiqing
Nie
,
Jinzhong
Qin
,
Timothy A.
Bird§, and
Xiaoxia
Li
¶
From the
Department of Immunology, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and
§ Amgen, Incorporated, Seattle, Washington 98101
Received for publication, November 27, 2002, and in revised form, December 19, 2002
 |
ABSTRACT |
The signaling pathway downstream of the mammalian
interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is
evolutionally conserved with that mediated by the
Drosophila Toll protein. Toll initiates its signal through
the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle
is highly homologous to members of the IL-1R-associated kinase (IRAK)
family in mammals. Recently, a novel Pelle-interacting protein called
Pellino was identified in Drosophila. We now report a
mammalian counterpart of Pellino, termed Pellino 1, which is required
for NF
B activation and IL-8 gene expression in response to IL-1,
probably through its signal-dependent interaction with
IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to
be intermediate, located between the IL-1 receptor complex and the TAK1
complex in the IL-1 pathway.
 |
INTRODUCTION |
Interleukin-1 (IL-1),1 a
major inflammatory cytokine, exerts its biological effects by
activating the transcription of various responsive genes (1). The
transcription factors activated by IL-1 include NF
B (Rel proteins),
AP1, and ATF (2-4). The IL-1 receptor belongs to the IL-1R/TLR
(Toll-like receptor) superfamily. Human TLRs have recently emerged
as key components in the generation of immune and inflammatory
responses because of their ability to recognize pathogen-associated
molecules (5). The signaling pathway downstream of mammalian IL-1R/TLR
is evolutionally conserved with that initiated by the
Drosophila Toll protein (5, 6). Drosophila Toll
controls dorsoventral axis formation in embryogenesis and has a role in
the anti-fungal immune response in the adult fly (6).
Upon IL-1 stimulation, the adaptor molecules MyD88 and Tollip are
recruited to the IL-1 receptor complex, which then recruits the
serine-threonine kinases IRAK4 and IRAK. IRAK is hyperphosphorylated, mediating the recruitment of TRAF6 to the receptor complex (7-12). IRAK-TRAF6 then leaves the receptor complex to interact with
pre-associated TAK1, TAB1, and TAB2 on the membrane. TAK1 and TAB2 are
phosphorylated on the membrane, which facilitates the formation and
translocation of the TRAF6-TAK1-TAB1-TAB2 complex from the membrane to
the cytosol. TAK1 is subsequently activated in the cytosol, leading to
the activation of I
B kinase (IKK). Activated IKK causes the
activation of NF
B through the phosphorylation and degradation of
I
Bs, which sequester NF
B prior to activation. Activation of TAK1
also results in the activation of mitogen-activated protein
kinases and c-Jun NH2-terminal kinase.
Drosophila Toll is activated by the endogenous ligand
Spaetzle, leading to the activation of Dif (Rel protein) through the degradation of Cactus, the Drosophila homologue of the
mammalian I
Bs, which sequester Dif prior to activation. Toll signals
through the adapter molecule Tube and the serine-threonine kinase Pelle (13). Although no mammalian counterpart has been found for Tube, Pelle
is highly homologous to IRAK4 and IRAK (7-10). Drosophila TRAF (dTRAF) has also been identified and shown to interact with Pelle
(14). Therefore, the Toll-Pelle-dTRAF-Cactus-Dif cascade is quite
analogous to the mammalian IL-1R/TLR-IRAK4/IRAK-TRAF6-I
B-NF
B pathway (14). However, the downstream signaling events from Pelle-dTRAF
to Cactus are not clear. When Pelle was used as bait in a yeast
two-hybrid screening, a novel Pelle-interacting protein called Pellino
was identified (15). Pellino was shown to only interact with the
catalytically active Pelle but not with a kinase-dead Pelle mutant,
suggesting that Pellino may play an important role in facilitating the
function of Pelle. In this study, we report the identification of
mammalian Pellino (termed Pellino 1) and its role in IL-1- mediated signaling.
 |
EXPERIMENTAL PROCEDURES |
Biological Reagents and Cell Culture--
Recombinant human
IL-1
was provided by the NCI, National Institutes of Health.
Anti-IRAK4 was a gift from Dr. Holger Wesche (Tularik, South San
Francisco, CA). Anti-TRAF6, anti-IRAK, and anti-IL-1R were from Santa
Cruz Biotechnology. Rabbit anti-TAK1, anti-TAB1, and anti-TAB2
polyclonal antibodies were kindly provided by Dr. Kunihiro Matsumoto
(16, 17). Anti-FLAG (M2) was from Sigma. HEK293-TK/Zeo cells,
I1A, and I3A (18) were maintained in Dulbecco's modified Eagle medium
supplemented with 10% fetal calf serum, penicillin G (100 µg/ml),
and streptomycin (100 µg/ml).
Recombinant Plasmids and Stable
Transfection--
pE-selectin-luc, an NF
B-dependent
E-selectin-luciferase (E-selectin-luc) reporter plasmid, was described
by Schindler and Baichwal (19). Dominant negative TAK1 (DNTAK1-K66W)
was a kind gift from Dr. Kunihiro Matsumoto (Nagoya University,
Nagoya, Japan). The vector pSUPER was obtained from Dr. Reuven Agami's
group (Center for Biomedical Genetics, Leiden, The
Netherlands). Pellino 1-pSUPER was constructed by cloning primers
derived from Pellino 1 (47-66 bp) according to the methods described
by Brummelkamp et al. (20). For stable transfections, 2 × 105 cells were seeded onto a 10-cm plate and
cotransfected the following day by the calcium phosphate method with 10 µg of each expression vector and 1 µg of pBabePuro. After 48 h, the cells were selected with 1 µg/ml puromycin until clones appeared.
Co-immunoprecipitation and Immunoblotting--
6 × 105 cells were seeded onto each 15-cm plate and transfected
the following day by the calcium phosphate method with 15 µg of
FLAG-tagged Pellino 1. After 48 h, cells untreated or treated with
100 units/ml of IL-1 were lysed in a Triton-containing lysis buffer
(0.5% Triton X-100, 20 mM HEPES, pH 7.4, 150 mM NaCl, 12.5 mM
-glycerophosphate, 1.5 mM MgCl2, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Cell extracts were
incubated with 1 µg of antibody or preimmune serum (negative control)
for 2 h, followed by a 2-h incubation with 20 µl of protein
A-Sepharose beads (pre-washed and resuspended in phosphate-buffered
saline at a 1:1 ratio). After incubation, the beads were washed four times with lysis buffer, separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and analyzed by immunoblotting.
Reporter Assays--
2 × 105 cells were
transfected by the same procedure as described above with 1 µg of
pE-selectin-luc, 1 µg of pSV2-
-gal, and 100 ng of each
expression construct. After 48 h, the cells were split onto two
35-mm plates and stimulated with IL-1 the next day for 4 h before
harvest. Luciferase and
-galactosidase activities were determined by
using the luciferase assay system and chemiluminescent reagents from
Promega (Madison, WI).
Gel Shift Assays--
An NF
B binding site
(5'-GAGCAGAGGGAAATTCCGTAACTT-3') from the IP-10 gene was used as a
probe (21). Complementary oligonucleotides, end-labeled with
polynucleotide kinase (Roche Molecular Biochemicals) and
-32P-labeled ATP, were annealed by slow cooling.
Approximately 20,000 cpm of probe were used per assay (22). Whole cell
extracts were used for the assay. The binding reaction was carried out
at 4 °C for 20 min in a total volume of 20 µl containing 20 mM Hepes buffer, pH 7.0, 10 mM KCl, 0.1%
Nonidet P-40, 0.5 mM dithiothreitol, 0.25 mM
phenylmethanesulfonyl fluoride, and 10% glycerol.
 |
RESULTS |
IL-1 Induces the Interaction of Pellino 1 with IRAK4 and
IRAK--
Human Pellino 1 cDNA, cloned from a human dermal
fibroblast cDNA library, encodes an open reading frame of 418 amino
acid residues (accession number NP_065702, Fig.
1a). To identify functionally important regions of Pellino 1, the mammalian Pellino 1 was aligned with those from other species (Fig. 1). There is a surprising degree of
similarity between the human Pellino 1 sequence and the homologous
protein from Caenorhabditis elegans, which share 44% amino acid identity and 53% similarity. The C-terminal portion of
Pellino 1 contains two invariant Cys-Gly-His triplets, two invariant
Cys-Pro-X-Cys motifs (where X is a hydrophobic
residue), and a conserved Cys-Pro-Val motif (Fig 1a),
reminiscent of the structure of the C3HC4 Ring finger subfamily of zinc
finger domains (23), which mediate diverse protein-protein interactions
of tumor suppressors, protooncogenes, and signaling molecules such as
TRAFs. Pellino 1 is ubiquitously expressed; it is expressed highly in
peripheral blood leukocytes, moderately in placenta, lung, liver,
kidney, spleen, thymus, skeletal muscle, and brain, and at a low level
in small intestine, colon and heart (Fig. 1b).

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Fig. 1.
Characterization of Pellino 1 cDNA and
expression pattern. a, deduced amino acid sequences of
Pellino 1 from human (Peli1.hu), mouse
(Peli1.mu), C. elegans (Peli1.ce), and
Drosophila melanogaster (Peli1.dm). The
motifs that resemble the structure of the C3HC4 Ring finger are
underlined. Identical residues are shaded
gray. b, Northern analysis of the
indicated mouse tissues using mouse Pellino 1 cDNA as a
probe.
|
|
We examined whether mammalian Pellino 1 interacts with IRAK and IRAK4,
which are the mammalian counterparts of Pelle (7, 9, 10, 24). Because
IRAK and IRAK4 are essential signaling components in the IL-1 pathway,
the interaction of Pellino 1 with IRAK and IRAK4 was studied upon IL-1
stimulation. Cell extracts prepared from HEK293 cells transfected with
FLAG-tagged Pellino 1, either untreated or stimulated with IL-1, and
immunoprecipitated with anti-FLAG M2 antibody followed by Western
immunoblotting using antibodies against IRAK and IRAK4. Interestingly,
Pellino 1 indeed forms an IL-1-dependent signaling complex
with the endogenous IRAK and IRAK4, strongly suggesting that it plays a
role in IL-1-mediated signaling (Fig.
2a).

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Fig. 2.
IL-1 signaling complexes. a
and b, IL-1-induced interaction of Pellino 1 with IL-1
signaling components. Cell extracts from HEK293 cells (293;
a) or HEK293 cells transfected with FLAG-Pellino 1 (293-Pellino 1; a and b) untreated
(0) or stimulated with IL-1 for the indicated times were
immunoprecipitated (IP) with M2 antibody
(anti-FLAG; a and b), anti-TRAF6
(b) and a control antibody (anti-p27,
1h*; a) followed by Western analyses with
antibodies against the FLAG tag on Pellino 1 (a and
b), IRAK (a), IRAK4 (a), TRAF6
(a and b), IL-1R (b), TAK1
(b), and TAB2 (b). c, model for
IL-1-mediated signaling pathway.
|
|
Pellino 1 Is Required for IL-1-induced NF
B Activation and IL-8
Gene Expression--
We used small interfering RNA (siRNA), a new gene
knock-down technology, to investigate the functional role of Pellino 1 in IL-1-mediated signaling pathways. Recently, a mammalian expression vector was developed that directs the synthesis of siRNA-like transcripts (pSUPER, suppression of endogenous RNA) (20). Pellino 1-pSUPER has been generated and stably transfected into HEK293 cells.
Northern analysis showed that 27% of the clones (clones 1, 3, 12, 16, and 18) transfected with Pellino 1-pSUPER had a 90% reduction of
Pellino 1 mRNA (Fig. 3a),
whereas the clones transfected with vector showed the same levels of
Pellino 1 mRNA (data not shown). As shown in Fig. 3b,
IL-1 induced much weaker NF
B activation in C-12 and C-16 (in which
Pellino 1 expression is knocked down; Fig. 3a) than in C-9
(a clone from the same transfection in which Pellino 1 expression is
not altered, Fig. 3a), strongly suggesting that Pellino 1 is
required for IL-1-mediated NF
B activation. The same expression
levels of NF
B were shown in these clones by immunoblotting with
antibodies against p65 and p50 (Fig. 3b). On the other hand,
the reduced expression of Pellino 1 had no effect on tumor necrosis
factor-induced NF
B activation, indicating that Pellino 1 is
specifically required for IL-1-dependent signaling. We also
examined the effect of reduced Pellino 1 expression on IL-1-induced
IL-8 gene expression, which was greatly reduced in C-12 and C-16 as
compared with C-9 (Fig. 3c), confirming that Pellino 1 is
indeed required for this IL-1-mediated signaling pathway.

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Fig. 3.
Reduced expression of Pellino 1 impairs IL-1
signaling. a, screening of clones with reduced
expression of Pellino 1. Total RNA from HEK293 cells stably transfected
with Pellino 1-pSUPER was analyzed by Northern procedure with a
Pellino1 gene-specific probe. B, NF B gel-shift assay.
Cell extracts from clones C-9, C-12, and C16, untreated or stimulated
with IL-1 (2, 4, and 8 h) and tumor necrosis factor (2 h), were
analyzed by NF B gel shift assay as described under "Experimental
Procedures." The same extracts were analyzed by immunoblotting with
anti-p65 and anti-p50. C, Northern analysis. RNA samples
from C-9, C-12, and C16, untreated or stimulated with IL-1 (4, 8, 12, 24 h), were analyzed by using IL-8 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNAs as probes.
|
|
IL-1-induced Pellino 1-IRAK4-IRAK-TRAF6 Is an Intermediate Complex
between IL-1 Receptor and TAK1 Complex--
Previous biochemical and
genetic studies are consistent with a model for IL-1-mediated signaling
(12) (Fig. 2c). To address the molecular mechanism by which
Pellino 1 functions in IL-1 signaling, we investigated whether Pellino
1 interacts with IRAK and IRAK4 in the receptor complex (Complex I) or
the TAK1-containing Complex II. Cell extracts prepared from HEK293
cells transfected with FLAG-tagged Pellino 1, either untreated or
stimulated with IL-1, were immunoprecipitated with anti-FLAG M2 (Fig.
2, a and b), anti-TRAF6 (Fig. 2b), and
anti-TAK1 (data not shown) and anti-TAB2 (data not shown)
antibodies followed by immunoblotting with antibodies against IRAK,
TRAF6, IL-1 receptor, TAK1, and TAB2. As shown previously (12), TRAF6
is present both in the receptor complex (interacting with IL-1R and
IRAK, Fig. 2, b and c) and the TAK1 complex
(interacting with TAK1 and TAB2, Fig. 2, b and
c). However, whereas Pellino 1 formed a complex with IRAK
(Fig. 2, a and b), IRAK4 (Fig. 2a), and TRAF6 (Fig. 2, a and b), it
co-immunoprecipitated with neither the IL-1 receptor (Fig.
2b) nor the TAK1 complex (TAK1 and TAB2; Fig. 2b,
and data not shown) upon IL-1 stimulation, suggesting that the
IL-1-induced Pellino 1-IRAK4-IRAK-TRAF6 is likely to exist as an
intermediate between the receptor complex (Complex I) and the
TAK1-containing complex (Complex II) (Fig. 2,
a-c).
Pellino 1 Functions Downstream of IRAK--
Because most of the
signaling components in the IL-1-mediated pathway are able to
constitutively activate NF
B upon overexpression, Pellino 1 was
examined for this activity. Pellino 1 in a mammalian expression vector
was co-transfected with NF
B-dependent E-selectin-luc into HEK293 cells followed by luciferase reporter assay. Pellino 1 can
activate the E-selectin promoter activity in a
dose-dependent manner (Fig.
4a). We have previously taken
a genetic approach to studying IL-1-dependent signaling
pathways through random mutagenesis, generating IL-1-unresponsive cell
lines lacking specific components of the pathways. Mutant cell line I1A
lacks both the IRAK protein and mRNA and is defective in
IL-1-induced NF
B and c-Jun NH2-terminal kinase
activation (18, 18, 25). As shown in Fig. 4a, whereas MyD88-mediated NF
B activation is completely abolished in IRAK deficient I1A cells, Pellino 1-mediated NF
B activation is intact in
these cells, indicating that Pellino 1 functions downstream of IRAK. On
the other hand, Pellino 1-induced NF
B activation was inhibited by a
dominant negative, kinase-inactive TAK1 mutant (TAK1 DN), indicating
that Pellino1 must function upstream of TAK1 (Fig. 4b). In
support of this conclusion, TAK1 can still activate NF
B in C-12,
where Pellino 1 expression is greatly knocked down by RNA interference
(Fig. 4c). Taken together, the above results support the
hypothesis that the Pellino 1-IRAK-IRAK4-TRAF6 signaling complex
functions between the receptor complex (Complex I) and the TAK1 complex
(Complex II).

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Fig. 4.
Pellino 1 functions upstream of TAK1 and
downstream of IRAK. a, Pellino 1 leads to NF B
activation in the IRAK-deficient I1A cells. FLAG-MyD88 (100 ng) and
FLAG-Pellino 1 (100 ng) expression constructs were co-transfected with
E-selectin-luc (100 ng) into HEK293 (WT, wild type) and the
IRAK-deficient I1A cells, followed by luciferase reporter assay. The
fold induction is relative to that in cells transfected with vector DNA
(100 ng) and reporter DNA (100 ng). Shown are the averages and standard
deviation from three independent experiments. b, kinase-dead
TAK1 mutant (TAK1DN) inhibits Pellino 1-induced NF B
activation. Increasing amounts of TAK1DN were co-transfected with
FLAG-Pellino 1 (0.5 µg) and E-selectin-luc (0.5 µg) into HEK293
cells, followed by luciferase reporter assay. The fold induction is
relative to that in cells transfected with only vector (3.5 µg) and
reporter DNA (0.5 µg). Shown are the averages and standard deviation
from three independent experiments. c, TAK1 leads to NF B
activation in clones transfected with Pellino 1-pSUPER. TAK1 (0.5, 1, and 3 µg of DNA) expression construct was co-transfected with
E-selectin-luc (100 ng) into C9 (with normal Pellino 1 expression) and
C12 (with reduced Pellino 1 expression), followed by luciferase
reporter assay. The fold induction is relative to that in cells
transfected with vector DNA (3 µg) and reporter DNA (100 ng). Shown
are the averages and standard deviation from three independent
experiments.
|
|
 |
DISCUSSION |
One possible function for Pellino 1 is linking TRAF6 to IRAK in
IL-1 signaling. However, several lines of evidence suggest that Pellino
1 is unlikely to be involved in the interaction between IRAK and TRAF6.
First, several TRAF6 binding sites have been identified in both human
and murine IRAK, and mutations in these sites abolish the ability of
IRAK to activate NF
B, suggesting that TRAF6 is likely to interact
with IRAK directly through these sites (26). Secondly, although our
previous results have clearly shown that TRAF6 is recruited to the IL-1
receptor through its interaction with IRAK upon IL-1 stimulation (Fig.
2, b and c) (12), Pellino 1 was not detected in
the IL-1-induced receptor complex (IL-1R-IRAK-TRAF6, Complex I; Fig. 2,
b and c). Therefore, it is unlikely that Pellino 1 is responsible for the IL-1-induced interaction between IRAK and TRAF6.
Previous studies have shown that the release of phosphorylated IRAK
from the receptor is essential for mediating downstream signaling
events in response to IL-1 (27). However, it is unclear how the
phosphorylated IRAK is released from the receptor. The fact that
Pellino 1 forms a complex with the phosphorylated IRAK but not with the
IL-1 receptor upon IL-1 stimulation (Fig. 2, a and
b) suggests that Pellino 1 may play an important role in facilitating the release of phosphorylated IRAK from the receptor.
It is important to note that Pellino 1 does interact with TRAF6 upon
IL-1 stimulation, whereas it does not interact with TAK1, suggesting
that Pellino 1 probably functions downstream of TRAF6 and upstream of
TAK1. In support of this, a dominant negative mutant of TAK1 inhibits
Pellino 1-mediated NF
B activation (Fig. 4b). The fact
that Pellino 1 can activate NF
B in IRAK-deficient cells (I1A)
suggests that Pellino 1 may have a direct impact on the activation of
TAK1. As a matter of fact, TAK1 is activated in I1A cells transfected
with Pellino 1 (data not shown). It is quite possible that Pellino 1 has a transient interaction with the TAK1 complex, which
could not be detected by the co-immunoprecipitation experiments
(Fig. 2c).
We have previously proposed a model for the IL-1 pathway (12). Taking
the above findings of Pellino 1 into consideration, we now propose a
modified model (Fig. 2c). Upon IL-1 stimulation, adapter
molecules MyD88 and Tollip are first recruited to the IL-1 receptor,
which in turn recruits IRAK4, IRAK, and TRAF6, resulting in the
formation of the receptor complex (Complex 1). During the formation of
Complex I, IRAK4 is activated, leading to the hyperphosphorylation of
IRAK, which creates an interface for the interaction of Pellino 1 with
the IRAK4-IRAK-TRAF6 complex. The formation of Pellino
1-IRAK4-IRAK-TRAF6 causes conformational changes in the receptor
complex (Complex I), which results in quick release of this
intermediate complex (Pellino 1-IRAK4-IRAK-TRAF6) from the receptor.
Through an unknown mechanism, the intermediate complex Pellino
1-IRAK4-IRAK-TRAF6 interacts with the membrane-bound pre-associated
TAK1-TAB1-TAB2, resulting in the formation of Complex II (TAK1 complex,
IRAK-TRAF6-TAK1-TAB1-TAB2). TRAF6-TAK1-TAB1-TAB2 (Complex III) is then
translocated from the membrane to the cytosol where TAK1 is activated,
eventually resulting in the activation of NF
B.
Although this study has shown the critical role of Pellino 1 in
IL-1-mediated signaling, it was recently reported that a related protein, Pellino 2, modulates IL-1 and lipopolysaccharide (LPS) signaling (28). At present, the relationship between Pellino 1 and
Pellino 2 and their specific functions in IL-1 signaling are still
unclear. One possibility is that each Pellino has its specific
interaction with IRAK and/or IRAK4. The elucidation of precise
functions of Pellino proteins in IL-1/TLR-mediated signaling will be an
exciting and important research area in the future.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hoger Wesche for the IRAK4
antibodies and Drs. Jun Ninomiya-Tsuji and Kunihiro Matsumoto for
dominant negative mutant TAK1 and antibodies against TAK1 and TAB2.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM 600020 (to X. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept.
of Immunology, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8706; Fax:
216-444-9329; E-mail: Lix@ccf.org.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M212112200
 |
ABBREVIATIONS |
The abbreviations used are:
IL-1, interleukin-1;
IL-1R, IL-1 receptor;
TLR, Toll-like receptor;
NF
B, nuclear factor
B;
IRAK, IL1R-associated kinase;
TRAF, tumor necrosis factor
receptor-associated factor;
TAK, transforming growth factor
-activated kinase;
TAB, TAK-1-binding protein;
Pellino 1, mammalian
Pellino;
HEK293, human embryonic kidney 293.
 |
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