ACCELERATED PUBLICATION
Double-stranded RNA Signaling by Toll-like Receptor 3 Requires
Specific Tyrosine Residues in Its Cytoplasmic Domain*
Saumendra N.
Sarkar,
Heather L.
Smith,
Theresa M.
Rowe, and
Ganes C.
Sen
From the Department of Molecular Biology, The Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, November 22, 2002, and in revised form, December 26, 2002
 |
ABSTRACT |
Double-stranded (ds) RNA, a common product of
viral infection, can induce transcription of many cellular genes,
including the 561 gene that encodes P56, a regulator of protein
synthesis. Here, we report that induction of the 561 mRNA by
exogenous dsRNA is mediated by Toll-like receptor 3 (TLR3), and it
requires no new protein synthesis. Because gene induction by dsRNA is
blocked by inhibitors of tyrosine kinases, we investigated the
potential roles of the five tyrosine residues present in the
cytoplasmic domain of TLR3 by their individual and combinatorial
mutations. Transfection assays, using a reporter gene driven by the 561 promoter, identified specific tyrosine residues to be essential for
TLR3 signaling. This conclusion was further validated in permanent cell
lines expressing tyrosine-mutant TLR3 proteins; in some of these cell
lines dsRNA failed to induce the 561 mRNA. Our results provide the
first demonstration of the importance of TLR3 cytoplasmic tyrosine
residues in dsRNA signaling.
 |
INTRODUCTION |
Double-stranded (ds)1
RNA is a potent regulator of gene expression in mammalian cells. It is
thought to be a critical viral gene product that modulates host
response in virally infected cells (1). In cell culture, the addition
of exogenous dsRNA causes rapid induction of more than a hundred genes,
many of which are also induced by type I interferons (IFN) and virus
infection (2). Among the most well studied dsRNA-induced genes is the human IFN-
gene. Its promoter is complex, and its induction requires the coordinate activation of members of the NF
B, AP-1, and IRF families of transcription factors (3, 4). In contrast, the human 561 gene has a simple promoter containing only IFN-stimulated response
elements and no
B sites (5). This gene can be induced strongly by
IFN or dsRNA. Moreover, microarray analyses have shown this gene to be
one of the most highly induced genes in virus-infected cells (2, 6, 7).
The encoded protein, P56, binds to the translation initiation factor
eIF-3 and inhibits protein synthesis (8). We have extensively used the
561 gene and its promoter to analyze the relevant dsRNA-signaling
pathway (5, 9). Our studies demonstrate that neither IFN nor the
Jak/STAT pathway of IFN signaling is required for 561 mRNA
induction by dsRNA (5). In contrast, the transcription factor IRF-3 is
critical for this induction process. Using mutant cells partially
responsive to dsRNA, we established conclusively that activation of
NF
B, AP-1, p38, or JNK is not required for IRF-3 activation and
consequent 561 mRNA induction. Thus, dsRNA uses a distinct IRF-3
pathway for activation of genes such as 561 (9).
Recently, the Toll-like receptor 3 (TLR3) has been shown to mediate
dsRNA response. Using TLR3-deficient cells and mice, Alexopoulou et al. (10) have demonstrated that TLR3 is required for
NF
B activation and mitogen-activated protein kinase activation by dsRNA. The TLR family of transmembrane receptors is an integral part of
our innate immune system. They recognize, with high specificity, diverse pathogen-associated molecular patterns and elicit intracellular signals to cause cellular inflammatory responses (11-13). Although there are partial overlaps among the genes induced by different TLRs,
their signaling pathways are thought to be distinct. However, the
complete signaling pathway has not been elucidated for any TLR. In this
study, we have investigated the role of TLR3 in the specific
dsRNA-elicited signaling pathway that causes induction of the 561 gene.
In the course of this investigation we have made the unexpected
observation that specific tyrosine residues of the cytoplasmic domain
of TLR3 are essential for this signaling pathway.
 |
MATERIALS AND METHODS |
Cells, Reagents, and Plasmids--
HEK293 cell line was
maintained in high glucose DMEM supplemented with 10% heat-inactivated
fetal calf serum, 2 mM L-glutamine, 50 units/ml
penicillin, and 50 µg/ml streptomycin. Anti-human TLR3 monoclonal
antibody was purchased from Biocarta (San Diego, CA). Cycloheximide and
genistein were purchased from Calbiochem.
The FLAG-TLR3 cDNA construct was obtained from Dr. Xiaoxia Li
(Cleveland Clinic Foundation). The TLR3 expression construct (FLAG-TLR3) we used in this study was constructed by removing the
original TLR3 signal peptide up to Phe 17 and replaced with the signal
peptide from pre-protrypsin, followed by a FLAG epitope tag (10) and
was cloned in pcDNA3 (Invitrogen). The
TIR-TLR3 construct
(
YYYYY) was made by PCR amplification of FLAG-TLR3 containing up to
amino acid 750 of TLR3. All the Tyr mutants of TLR3 were generated by
the mega-primer PCR method using appropriate primers and FLAG-TLR3 as
the template. Mutations were confirmed by sequencing.
Development of 293/TLR3 and 293/TLR3-mutant Cell
Lines--
HEK293 cells were seeded into 100-mm dishes and transfected
using Lipofectamine 2000 (Invitrogen) with 6 µg of the appropriate plasmid DNA. Cells were selected in DMEM containing 530 µg/ml (400 µg/ml active) G418 (Research Products International, Mt. Prospect,
IL). Individual colonies were picked, expanded, and confirmed for TLR3
expression by Western blotting with anti-TLR3 monoclonal antibody
(final concentration: 2 µg/ml). For Western blotting, 2.5 × 106 cells were lysed in lysis buffer containing 300 mM NaCl, 20 mM Tris-Cl (pH 7.5), 5 mM
-mercaptoethanol, 0.2% Triton X-100, 10% glycerol,
and protease inhibitors (complete EDTA-free protease inhibitor tablets,
Roche Molecular Biochemicals). 150 µg of total protein lysates were
used for Western analysis. Stable cell lines were propagated and
maintained in complete DMEM supplemented with 530 µg/ml G418.
Ribonuclease Protection Assay (RPA)--
RNA was isolated with
RNA-Bee (Tel-Test, Friendswood, TX). RPAs were performed with the RPA
III kit (Ambion, Austin, TX) following the manufacturer's protocol.
The 561 and actin RPA probes had been described previously (9). For
each sample 20 µg of total RNA was used for RPA. Protected mRNA
levels were visualized by autoradiography as well as quantified using
PhosphorImager (Amersham Biosciences).
561-Luciferase Transient Transfection Assay--
The
561-luciferase reporter construct contains the
3 to
654 nucleotides
of the P56 promoter (14) cloned into the
SacI/HindIII restriction sites of the pGL3B
vector (Promega, Madison, WI). The pRL-SV40 vector coding for Renilla
luciferase (Promega) was used as the internal control for normalization
of transfection efficiency. HEK293 cells were transfected in six-well
dishes using 5 µl of Lipofectamine 2000 (Invitrogen) with 50 ng of
561-luciferase, 5 ng of pRL-SV40, and 200 ng of wt TLR3 or mutant TLR3
expression vectors. After 6 h, transfection medium was
removed, and cells were washed with phosphate-buffered saline
and returned to complete medium for overnight incubation. The
cells were then treated for 6 h, with (100 µg/ml)
poly(I)·poly(C) as described previously (9). Luciferase assay was
done using the dual luciferase reporter assay system (Promega)
following manufacturer's protocol. For each experiment, transfections
and luciferase assays were done in triplicate, and each experiment was
repeated three times. Fold induction of 561-luciferase was calculated
and averaged against uninduced control after normalizing luciferase
activity with respect to Renilla luciferase. Similar levels of wt and
mutant TLR3 expression in the transfected cells were confirmed by
Western blotting of the extracts.
Detection of Phosphotyrosines in TLR3--
4 × 107 cells were treated with poly(I)·poly(C) for 1 h
or left untreated for control. Lysates were made by incubating cell suspensions on ice for 30 min in lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 0.5% Triton X-100, 10 mM NaF, 2 mM dithiothreitol, 1 mM Na3VO4, 12.5 mM
-glycerophosphate, and protease inhibitors. Equal amounts of
proteins were immunoprecipitated with 20 µl of anti-FLAG M2 antibody
agarose (Sigma) for 4 h. Following four washes with the lysis
buffer, beads were boiled in 1× SDS-PAGE loading buffer and analyzed
on SDS-PAGE. Phosphotyrosines were detected by Western blotting with
PY20 antibody (1:2000, Pharmingen). Blots were stripped and reprobed
with anti-FLAG M2 antibody for detecting TLR3.
 |
RESULTS |
TLR3-dependent Induction of 561 mRNA--
The
human kidney cell line 293 does not express TLR3, and addition of dsRNA
to its culture medium did not cause induction of the 561 mRNA, but
the same mRNA was induced strongly in the 293 cell line expressing
TLR3 (Fig. 1A). 561 mRNA
was not detectable in untreated 293/TLR3 cells, but it was rapidly
induced after dsRNA treatment. This induction was direct and did not
require the synthesis of any new protein. 561 mRNA was induced by
dsRNA equally well in the presence of cycloheximide, a potent inhibitor of protein synthesis (Fig. 1B).

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Fig. 1.
TLR3-mediated 561 mRNA induction by
dsRNA. The levels of 561 mRNA (filled arrow) and
actin mRNA (open arrow) were measured by RPA.
A, 293 and 293/TLR3 cells were left untreated ( ) or
treated with poly(I)·poly(C) for 6 h (+). RPA was performed to
measure 561 mRNA and actin mRNA levels. B, 293/TLR3
cells were pretreated with 50 µg/ml cycloheximide for 30 min, where
indicated, before treating with poly(I)·poly(C) for 6 h, in the
presence of cycloheximide. RPA was done to measure 561 mRNA
induction.
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Role of Specific Tyrosine Residues of TLR3 in dsRNA
Signaling--
There is information in the literature demonstrating
the dependence of dsRNA signaling on protein-tyrosine kinase activity (15). We observed that the induction of 561 mRNA by dsRNA was blocked by genistein, an inhibitor of tyrosine kinases (data not shown). This observation suggested to us that tyrosine residues present
in the cytoplasmic domain of TLR3 might be required for its ability to
transmit dsRNA-elicited signals. The putative cytoplasmic domain of
TLR3 has 130 residues, out of which five are tyrosines (Fig.
2A). One tyrosine, tyrosine
733, is situated very close to the transmembrane domain and another,
tyrosine 858, is located distally. Three other tyrosine residues,
tyrosines 756, 759, and 764, are clustered in a region about 30 residues away from the transmembrane domain. We wanted to examine the
roles of these five tyrosine residues in TLR3 signaling by mutating
them conservatively to phenylalanine residues individually or in
combination.

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Fig. 2.
Requirement of specific Tyr residues in the
cytoplasmic domain of TLR3 for the induction of 561-luciferase by
dsRNA. A, positions of the five tyrosine residues in the
cytoplasmic domain of TLR3 are shown. B, these Tyr were
mutated individually or in combinations to Phe to study their roles in
TLR3-mediated induction of 561-luciferase expression by dsRNA. 293 cells were co-transfected with expression vectors of wt or mutant TLR3,
the 561-luciferase reporter gene, and the normalization control
pRL-SV40. Cells were treated with dsRNA, where indicated, for 6 h
before measuring luciferase levels. On the left, the
mutational statuses of the five Tyr residues are shown. All samples,
except one, were treated with dsRNA. The bars on the
right show normalized -fold inductions of luciferase
activity. The error bars are from three independent
experiments, each done in triplicates. C, the relative
importance of Tyr residues in signaling is shown. Tyr759
plays the most crucial role followed by the lesser but equally
important roles of Tyr733 or Tyr858.
Tyr756 and Tyr764 have the least important
roles.
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A reporter assay was developed to test the function of these mutant
proteins. For this purpose, a luciferase reporter gene driven by the
promoter of the 561 gene was co-transfected with an expression vector
of wt or mutant TLR3 to 293 cells, and the level of luciferase
expression was measured after dsRNA treatment. That wt and mutant TLR3
proteins were expressed to similar levels was ensured by Western blot
analysis of transfected cell extracts (data not shown). When wt TLR3
was used, there was little expression of luciferase in cells not
treated with dsRNA (Fig. 2B, line 1). However,
the expression level was elevated about 20-fold when cells were treated
with dsRNA (Fig. 2B, line 2). A mutant TLR3, from
which most of the cytoplasmic domain had been deleted (residues 751-860 deleted), failed to signal even in the presence of dsRNA (Fig.
2B, line 3). The above characteristics of the
reporter assay established its validity and the obvious need for the
cytoplasmic domain of TLR3 for its ability to signal.
A critical experiment of this series is shown in line 4 of
Fig. 2B. When all five Tyr residues were mutated to Phe, the
receptor failed to signal even in the presence of dsRNA. This result
demonstrated an absolute need for the presence of the Tyr residues in
the cytoplasmic domain of TLR3 for its ability to signal. The 5F
mutant, with all five residues mutated, was subsequently used as the
starting material for restoring one or more Tyr residues at a time.
Restoration of only Tyr731 had no effect (line
5, Fig. 2B). On the other hand, restoration of
Tyr759 by itself elicited a small, but significant, level
of signal (line 6, Fig. 2B). The 561-luciferase
activity was restored fully when amino acid 858 was restored back to
Tyr as well (line 7, Fig. 2B), although the other
three residues (733, 756, 764) remained mutated. Other combinations
containing 759 and another Tyr residue and three Phe residues had
intermediate activities (lines 8-10, Fig. 2B).
In contrast, if the two Tyr residues did not include the 759 moiety,
the mutant proteins were completely inactive (line 11, Fig.
2B and data not shown). The same was true for proteins containing three out of five Tyr residues restored. The proteins were
active only if the 759 residue was a Tyr (lines 12-14, Fig. 2B). Finally, a series of mutants, which contained single
Tyr to Phe mutations, was tested. Mutants Y756F (line 16,
Fig. 2B) and Y764F (line 18, Fig. 2B)
were completely active, whereas Y733F (line 15, Fig.
2B) and Y858F (line 19, Fig. 2B) were
slightly less potent than the wt protein. As expected, the Y759F mutant (line 17, Fig. 2B) was the least active, although
about 20% activity remained. The above series of reporter assays
strongly indicated that the Tyr residues of the TLR3 cytoplasmic domain
are essential for dsRNA signaling to the 561 gene. Moreover, there is a
distinct hierarchy among the five Tyr residues with respect to their
contributions to signaling (Fig. 2C). The Tyr at 759 is the
most critical residue, its mutation to Phe strongly diminished the
ability of the receptor to signal (line 17, Fig.
2B).
Although the above results were quite convincing, they were generated
by transient reporter assays, which sometimes do not reflect completely
the properties of the resident gene. For this reason, we established
new 293 cell lines expressing either the wt or various mutant TLR3
proteins. Clones expressing comparable levels of the wt and a mutant
protein were matched and used for testing induction of the 561 mRNA
in response to dsRNA (Fig. 3). The wt-18
clone and the 5F-24 expressed similar levels of the wt and the five Tyr
to Phe mutant TLR3 proteins, respectively (Fig. 3A). As
expected, 561 mRNA was strongly induced by dsRNA in the wt-18
cells, but no induction could be detected in the 5F-24 cells (Fig.
3B). Another set of cell lines, wt-11, FFYFY-9, YFFFY-9 and
759F-30, expressed the wt or the mutant proteins to similar levels
(Fig. 3C). Again, the wt-11 clone strongly expressed 561 mRNA in a dsRNA-dependent manner, as did the cells
expressing the FFYFY mutant. In contrast, cells expressing the YFFFY or
the 759F mutants were unresponsive (Fig. 3D). These results
provided definitive evidence for the need of the cytoplasmic Tyr
residues of TLR3, especially the one at 759, for mediating
dsRNA-elicited signaling to cause transcriptional induction of the 561 mRNA.

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Fig. 3.
Involvement of cytoplasmic Tyr residues of
TLR3 in dsRNA induced 561 mRNA induction. Stable cell lines
expressing wt and mutant TLR3 proteins were made as described under
"Materials and Methods." The 5F mutant had all five Tyr mutated to
Phe, and the 759F had only the 759 residue mutated. The other two
mutants had three mutations indicated by their names. The number after
each name indicates the cell clone number. A and
C, total protein lysates were made from each clonal cell
lines and analyzed for TLR3 expression by Western blotting.
B and D, induction of the 561 mRNA in
response to dsRNA was tested in these lines by RPA. The filled
arrow indicates protected 561 mRNA, whereas the actin mRNA
control is shown by the open arrow.
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Ligand-induced Tyrosine Phosphorylation of TLR3--
Finally, we
wanted to examine whether the cytoplasmic Tyr residues of TLR3 were
phosphorylated. Results presented in Fig. 4 show that the wt TLR3 gets
Tyr-phosphorylated in its cytoplasmic domain. wt-18 and 5F-24 cells
stably expressing the wt and the 5F mutant TLR3 proteins, respectively
(Fig. 3), were treated with dsRNA for 1 h, and TLR3 proteins were
immunoprecipitated and Western blotted with anti-phosphotyrosine
antibody. A phosphotyrosine-specific protein band appeared at the TLR3
position, only after dsRNA treatment of wt-18 cells. (Fig.
4A, lane 3), but not in 5F-24 cells (Fig. 4A, lane 4). A slightly faster migrating
phosphorylated protein of unknown identity was present in all samples.
Comparable amounts of TLR3 proteins were immunoprecipitated from all
extracts containing TLR3 (Fig. 4B). These results
demonstrated that TLR3 undergoes Tyr phosphorylation in its cytoplasmic
domain upon dsRNA stimulation of the cells.

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Fig. 4.
Phosphorylation of cytoplasmic tyrosine
residues of TLR3. 293 cells stably expressing FLAG-tagged wt TLR3
(wt-18) and 5F mutant TLR3 (5F-24) were treated with dsRNA or left
untreated. Cell lysates were immunoprecipitated with anti-FLAG antibody
and Western blotted with anti-phosphotyrosine antibody (A)
followed by anti-FLAG antibody (B). Lane 1,
untreated 293 cells; lane 2, wt-18 cells; lane 3,
wt-18 cells treated with poly(I)·poly(C) for 1 h; lane
4, 5F-24 cells treated with poly(I)·poly(C) for 1 h.
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 |
DISCUSSION |
Transcriptional induction by dsRNA of the 561 gene and other
IFN-stimulated genes is mediated by members of the IRF family, primarily IRF-3 (1, 16). This signaling pathway is independent of
dsRNA-mediated activation of I
B kinase, JNK, or p38 kinases, and it does not require the activities of transcription factors such as
NF
B or AP-1 (9). Alexopoulou et al. (10) have shown that
the 293 cells are devoid of the latter responses to dsRNA unless TLR3
is expressed in them by transfection. Here, we have established the
same requirements for another dsRNA-elicited signaling pathway, which
leads to 561 gene activation. To validate direct induction of the 561 gene by dsRNA in 293 cells, we performed the cycloheximide experiment
(Fig. 1B). Since the 561 gene and other genes of this family
can also be induced by type I IFN, there was a possibility that the
observed induction of 561 mRNA was mediated by intermediate IFN
synthesis. However, if that were the case, the induction would have
been inhibited by blocking new protein synthesis. In other cell types,
such as HT1080 and GRE using genetic and biochemical approaches, we
have previously shown that the 561 gene can be induced by dsRNA without
any involvement of IFNs or their receptors (5, 9). Here, the same
conclusion was formally established for 293/TLR3 cells as well. Thus,
the 561 gene can be considered as a primary response gene for dsRNA in
these cells. The fact that tyrosine kinase inhibitors could block 561 mRNA induction led us to study the role of Tyr residues of TLR3 in
dsRNA signaling. Our study has established a possible connection
between that susceptibility and TLR3 tyrosine residues.
The transient reporter assay shown in Fig. 2 has been used by us before
in other cells (5, 7). In the 293 cells it was quite robust and
reproducible with a low background. More importantly, it was totally
dependent on the expression of transfected TLR3, thus offering us a
convenient and rapid assay for mutant TLR3 functions. Taking advantage
of this assay, we could establish the relative functional importance of
the five tyrosine residues of the cytoplasmic domain of TLR3. It was
clear that Tyr759 was the most critical residue, but for
maximal activity of the promoter, the presence of another Tyr was
needed. For this accessory function, the most membrane-proximal (733)
or the most distal (858) Tyr residue was most effective, whereas the
two other Tyr residues present near the Tyr759 (756 and
764) were less effective. The conclusions drawn from the reporter
assays were confirmed by examining induction of the resident 561 gene
in cell lines derived from 293 cells that expressed wt or mutant TLR3
proteins. Cells expressing the 5F and the YFFFY mutants were totally
unresponsive, whereas those expressing the FFYFY mutant were as
responsive as the wt cells (Fig. 3). Cells expressing the Y759F mutant
were also unresponsive. The Y759F mutant, however, had some residual
activity in the transient reporter assay (Fig. 2B,
line 17). This minor difference could be attributed to high
overexpression of the transfected proteins in the transient assays. It
could also be a genuine difference between the responses of the
chromosomal gene and the transfected reporter gene driven by an
arbitrarily truncated promoter. Thus, the lack of response of the
resident 561 gene in cells expressing the Y759F protein is the more
physiologically relevant result.
We observed Tyr phosphorylation of wt-TLR3, but not of 5F-TLR3, in
response to dsRNA treatment (Fig. 4). We do not know at this time which
specific Tyr residues get phosphorylated nor do we know how rapidly it
happens. It is tempting to speculate that the phosphotyrosine moieties
serve as the docking sites for protein kinases or adaptor proteins,
thereby starting a cascade of signaling events leading to activation of
the relevant transcription factors. Drawing upon the knowledge of
growth factor receptors, it is also conceivable that different tyrosine
residues of TLR3 cytoplasmic domains attract different protein partners
and hence initiate different independent signaling pathways (17).
Extensive genetic and biochemical investigations will be required in
the future to examine these possibilities. Irrespective of the
mechanism, it is clear from this study that cytoplasmic tyrosine
residues and their phosphorylation play an essential role in TLR3
signaling. This observation is highly significant, because tyrosine
phosphorylation was not thought to be connected to the primary
signaling pathway of any member of the TLR family. The only exception
is TLR2, which has recently been shown to contain phosphotyrosine
residues in its cytoplasmic domain (18). In that case, however, the
critical tyrosine residues have not been identified, but presumably
they are required for the assembly of the signaling complex.
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ACKNOWLEDGEMENTS |
We thank Xiaoxia Li, Joe DiDonato, Bryan
Williams, Kristi Peters, and Chris Elco for reagents and
helpful discussions; Srabani Pal for technical assistance; and Karen
Toil for secretarial assistance.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants CA62220 and CA68782.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 Molecular
Biology/NC20, The Lerner Research Inst., The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-0636; Fax: 216-444-0513; E-mail: seng@ccf.org.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.C200655200
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ABBREVIATIONS |
The abbreviations used are:
ds, double-stranded;
TLR3, Toll-like receptor 3;
IFN, interferon;
RPA, ribonuclease protection assay;
poly(I)·poly(C), polyinosinic acid
polycytidylic acid;
JNK, c-Jun NH2-terminal kinase;
DMEM, Dulbecco's modified Eagle's medium;
wt, wild type.
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.