Dual-specificity phosphatase 5 acts as an anti-inflammatory regulator by inhibiting the ERK and NF-κB signaling pathways

Although dual-specificity phosphatase 5 (DUSP5), which inactivates extracellular signal-regulated kinase (ERK), suppresses tumors in several types of cancer, its functional roles remain largely unknown. Here, we show that DUSP5 is induced during lipopolysaccharide (LPS)-mediated inflammation and inhibits nuclear factor-κB (NF-κB) activity. DUSP5 mRNA and protein expression increased transiently in LPS-stimulated RAW 264.7 cells and then returned to basal levels. DUSP5 overexpression in RAW 264.7 cells suppressed the production of pro-inflammatory tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), whereas knockdown of DUSP5 increased their expression. Investigation of two major inflammatory signaling pathways, mitogen-activated protein kinase (MAPK) and NF-κB, using activator protein-1 (AP-1) and NF-κB reporter plasmids, respectively, showed that NF-κB transcription activity was downregulated by DUSP5 in a phosphatase activity-independent manner whereas AP-1 activity was inhibited by DUSP5 phosphatase activity towards ERK,. Further investigation showed that DUSP5 directly interacts with transforming growth factor beta-activated kinase 1 (TAK1) and inhibitor of κB (IκB) kinases (IKKs) but not with IκBα. DUSP5 binding to IKKs interfered with the association of TAK1 with IKKs, suggesting that DUSP5 might act as a competitive inhibitor of TAK1-IKKs association. Therefore, we propose that DUSP5 negatively regulates ERK and NF-κB in a phosphatase activity-dependent and -independent manner, respectively.

Phosphorylation of serine, threonine, or tyrosine residues in proteins is a typical post-translational modification in eukaryotes that is a critical part of signal transduction pathways involved in important cellular processes such as cell differentiation, proliferation, apoptosis, gene expression, cytoskeletal function, and immunological signaling 1 . Protein phosphorylation is regulated by the equal and balanced action of protein kinases and phosphatases in mammalian cells 2 .
Macrophages are innate immune cells activated during microbial infection and are vital mediators of innate immune responses such as phagocytosis, antigen presentation, and secretion of cytokines, chemokines, and several other factors 3 . Macrophages stimulated by lipopolysaccharide (LPS) release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-12, monocyte chemotactic protein-1, interferon-gamma, and IL-10 through complex signaling mechanisms 4 . Stimulated macrophages and dendritic cells localized to affected tissues recognize pathogen-associated molecular patterns via specific receptors, including Toll-like receptors and nucleotide-binding oligomerization domain-containing proteins 5,6 . Then, adaptor proteins, including myeloid differentiation factor 88 (MYD88) and Toll/IL-1 receptor domain-containing adapter protein inducing interferon-β (TRIF), in turn activate the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways 7 . MAPKs induce cytokine gene expression by promoting transcription factors such as activator protein-1 (AP-1), which enhance the stability of cytokine and chemokine mRNAs 8

DUSP5 expression is transiently induced by LPS in macrophages.
Several signaling pathways in innate immune cells are activated by a protein phosphorylation cascade that leads to synthesis of pro-inflammatory cytokines that mobilize the immune system to combat LPS, endotoxins derived from pathogenic gram-negative bacteria 25 . During inflammation, phosphorylation of signaling components is regulated by phosphatases induced by LPS stimulation 26,27 . Since several PTPs are induced or suppressed by LPS in order to control protein phosphorylation during inflammation in macrophages 12,14-17 , we performed RT-PCR with RNA samples prepared from RAW 264.7 cells stimulated with LPS for 1 or 3 h, using gene-specific primers against previously untested PTP genes (Table 1). DUSP1 primers were used as a positive control, since DUSP1 expression is known to be induced by LPS 28 . Of the PTP genes tested, DUSP5 expression increased upon exposure to LPS, whereas the expression of the other PTPs did not change (Fig. 1a). The list of PTPs analyzed in this study and in previous studies is shown in Table 2. DUSP5 mRNA expression was induced within 1 h of LPS stimulation, which then began to decline by 3 h of LPS stimulation. Using quantitative real-time polymerase chain reaction (qRT-PCR), we confirmed the kinetics of DUSP5 mRNA expression changes after LPS treatment. DUSP5 mRNA expression increased when the cells were treated with LPS for 1 h and then slowly returned to near-basal levels by 24 h (Fig. 1b). In addition, when DUSP5 protein expression levels were analyzed by immunoblotting using an anti-DUSP5 antibody, the protein expression pattern corresponded to the mRNA expression pattern, although it was slightly delayed (Fig. 1c). Similar to DUSP5, DUSP1 was induced at early time points following LPS exposure and then returned to basal levels after 2 h of LPS treatment, as reported previously 28 . Endogenous DUSP5 was immunoprecipitated from LPS-treated RAW 264.7 and its phosphatase activities were measured using OMFP as a substrate. DUSP5 phosphatase activity was found to be increased in proportion to the increase in its protein expression (Fig. 1d). These results suggest that DUSP5 expression and its activity are induced by LPS treatment at an early stage.
DUSP5 inhibits TNF-α and IL-6 production. Since LPS induces DUSP5 expression in RAW 264.7 cells, DUSP5 might be involved in the regulation of pro-inflammatory cytokines. To investigate the effect of DUSP5 on cytokine production, we first analyzed the levels of TNF-α and IL-6, which are major inflammatory cytokines induced in macrophages upon exposure to endotoxins such as LPS 29,30 . To examine the effect of DUSP5 on TNF-α and IL-6 production, RAW 264.7 cells were transfected with a mammalian expression vector  Protein expression levels were analyzed by immunoblotting and quantified by scanning the immunoblots and analyzing the scans using LabWorks software.
containing FLAG-tagged DUSP5 wild type (WT) or the catalytically inactive C263S mutant for 32 h and then stimulated with 1 μg/ml LPS for 16 h prior to assessment of TNF-α and IL-6 production by ELISA. Compared to LPS-treated cells, both DUSP5 WT-and C263S mutant-expressing cells produced reduced levels of TNF-α and IL-6 ( Fig. 2a and b). However, the inhibitory effect of DUSP5 C263S mutant on cytokine production was weaker than that of DUSP5 WT, which suggests that DUSP5 regulates both TNF-α and IL-6 in a phosphatase activity-dependent and -independent manner. To further confirm that the production of TNF-α and IL-6 is regulated by DUSP5, DUSP5-specific siRNAs were transfected into RAW 264.7 cells to knock down DUSP5 gene expression. Reduced levels of DUSP5 expression after transfection with DUSP5-specific siRNAs (#1 and #2) were confirmed by immunoblotting (Supplementary Figure 1). Following transfection with either scrambled control siRNA or DUSP5-specific siRNAs (#1 and #2), RAW 264.7 cells were treated with a low dose of LPS (0.1 μg/ml) for 1 h to avoid saturation of TNF-α and IL-6 production, and the effects of DUSP5 knockdown were then determined by measuring the TNF-α and IL-6 levels in the growth medium. As shown in Fig. 2c,d, DUSP5 knockdown enhanced LPS-induced TNF-α and IL-6 production in RAW 264.7 cells. Furthermore, TNF-α and IL-6 levels were increased in the absence of LPS when DUSP5 was knocked down. Taken together, these results suggest that DUSP5 inhibits the production of TNF-α and IL-6 in RAW 264.7 macrophages.
DUSP5 regulates NF-κB as well as ERK1/ 2 signal transduction. Pro-inflammatory cytokines are regulated by two major signaling pathways, MAPK and NF-κB. During inflammation, inflammatory gene expression is induced by the activation of specific transcription factors such as AP-1 and NF-κB. The signal transduction cascade after pathogenic stimulation results in the activation of parallel kinase cascades that regulate AP-1 and NF-κB 25 . We therefore carried out luciferase activity-based reporter assays to investigate whether DUSP5 regulates AP-1 and NF-κB transcription activity. An AP-1-or NF-κB-Luc reporter plasmid was co-transfected with the FLAG-tagged DUSP5 WT or C263S plasmid into HEK 293 cells. Transfected cells were then treated with PMA to stimulate AP-1 and NF-κB activity. The PMA treatment was sufficient for stimulating the reporter genes and transient expression of DUSP5 WT resulted in decreased transcriptional activity of AP-1 and NF-κB in a dose-dependent manner (Fig. 3a,b). Interestingly, DUSP5 C263S inhibited the PMA-induced NF-κB transcriptional activity, whereas the mutant failed to inhibit AP-1 activity, suggesting that DUSP5 regulates NF-κB transcription in a phosphatase activity-independent manner, whereas regulation of AP-1 by DUSP5 is dependent on its phosphatase activity (Fig. 3b), which is reminiscent of the data on the regulation of TNF-α and IL-6 production by DUSP5 C263S mutant, as shown in Fig. 2a   confirmed that DUSP5 dephosphorylates the TXY motifs of ERK1/2 but not p38 or JNK in RAW 264.7 cells using phospho-specific antibodies. Immunoblotting analysis of lysates from RAW 264.7 cells transfected with FLAG-tagged DUSP5 WT or C263S plasmids followed by stimulation with LPS showed that the endogenous phospho (p)-ERK1/2 level was markedly reduced in DUSP5 WT-transfected cells compared to that in cells transfected with the C263S mutant (Supplementary Figure 2). Under the same conditions, the levels of phosphorylated JNK and p38 were unchanged. To confirm that DUSP5 expression affects the phosphorylation of IκBα, DUSP5 was knocked down by transfection with DUSP5 siRNA #1 into RAW 264.7 cells. DUSP5 knockdown enhanced the phosphorylation of IκBα at Ser-32/36 and degradation of IκBα, compared to that in cells transfected with the non-targeting control siRNA (Fig. 4a). ERK1/2 phosphorylation was enhanced by DUSP5 knockdown, as expected. These results suggest that DUSP5 regulates the levels of p-IκBα as well as p-ERK1/2 in LPS-stimulated RAW 264.7 cells.
Unlike ERK1/2, NF-κB has not been reported as a target of DUSP5. Therefore, we investigated how DUSP5 regulates NF-κB activity. Since phosphorylation of IκBα at Ser-32/36 is the most well-known process leading to ubiquitin-mediated degradation of IκBα and release of NF-κB for nuclear translocation 31,32 , we examined the levels of p-IκBα (Ser-32/36) in DUSP5-transfected cells by immunoblotting analysis. Similar to the result in Fig. 3b, both DUSP5 WT and C263S reduced the phosphorylation levels of IκBα Ser-32/36 and inhibited the degradation of IκBα in a dose-dependent manner (Fig. 4b). To clarify the mechanism of action of DUSP5 on regulation of NF-κB signal transduction, the phosphorylation levels of signaling kinases upstream of IκBα, including IKKα/β and TAK1, in the presence of DUSP5 was investigated. As shown in Fig. 4b, phosphorylation at Ser-176/180 of IKKα/β, a kinase complex directly upstream of IκBα, was also decreased by DUSP5 WT or C263S expression. However, DUSP5 WT-and C263S-transfected cells did not result in altered phosphorylation levels at Thr-184/187 Cell lysates were subjected to immunoblotting using an anti-FLAG antibody for the detection of DUSP5. The results represent three independent experiments. *p < 0.05 versus LPS-treated cells transfected with empty vector (Student's t-test). After transfection with control or DUSP5 siRNAs (#1 and #2), cells were treated with 0.1 μg/ml LPS for 1 h, and the levels of TNF-α (c) and IL-6 (d) were measured by ELISA. The results represent mean data from three independent experiments. *p < 0.05 versus LPS-treated or untreated cells transfected with control siRNA (Student's t-test).
of TAK1 that phosphorylates and activates IKKs. These results imply that DUSP5 might regulate the NF-κB signaling pathway by acting on TAK1 independently of phosphatase activity.
The regulatory roles of DUSP5 in the NF-κB signaling pathway were confirmed by employing DUSP5 WT and KO mouse embryonic fibroblasts (MEFs). We examined the phosphorylation levels of NF-κB signal transduction molecules, including IκBα, IKKα/β, and TAK1 (Fig. 4c). Treatment with LPS for stimulation of NF-κB signal transduction did not result in any response from the cells. Therefore, MEFs were treated with TNF-α to stimulate NF-κB signal transduction because TNF-α-induced NF-κB activation is mediated by activation of the TAK1/ IKK/IκBα axis. The phosphorylation levels of IκBα, IKKα/β, and TAK1 were enhanced by TNF-α stimulation in both DUSP5 WT and KO cells. However, DUSP5 KO cells showed a greater increase in the fold induction of IκBα and IKKα/β phosphorylation than that in the DUSP5 WT cells. IκBα levels decreased due to degradation of phosphorylated IκBα in DUSP5 KO cells upon TNF-α stimulation, implying that DUSP5 expression is critical for the activation of NF-κB signal transduction via inhibition of IKKα/β and subsequent IκBα phosphorylation and degradation. However, no difference in TNF-α-induced phosphorylation of TAK1 between DUSP5 WT and KO cells was obvious. These results suggest that DUSP5 expression is critical for the negative regulation of NF-κB signal transduction by either dephosphorylating IKKα/β or by physically inhibiting TAK1.
Since our data suggest that DUSP5 acts as a regulator of NF-κB signaling in the cytoplasm, we next investigated the subcellular localization of endogenous DUSP5 in LPS-stimulated RAW 264.7 cells. As shown in Fig. 4d, DUSP5 expression was detected in both the cytoplasm and the nucleus. In addition, treatment with LPS resulted in significantly increased expression of DUSP5 in both the cytoplasmic and nuclear fractions. Although nuclear localization of DUSP5 has been reported 22 , our data provide evidence that DUSP5 can also exist in the cytoplasm and that DUSP5 expression in both the cytoplasm and the nucleus is upregulated upon LPS stimulation. DUSP5 physically interacts with TAK1 and IKKα/β and blocks the association of TAK1 with IKKα/β.
As described above, DUSP5 inhibits the phosphorylation of IκBα and IKKα/β in a phosphatase activity-independent manner. Furthermore, DUSP5 had no effect on TAK1 phosphorylation. These data led us to examine the regulatory mechanism of DUSP5 by investigating the interaction partners of DUSP5 in the NF-κB signaling pathway. Since endogenous DUSP5 was induced upon LPS stimulation in RAW 264.7 cells, we carried out co-immunoprecipitation using either pre-immune IgG or anti-DUSP5 antibody with total cell lysates obtained from LPS-stimulated RAW 264.7 cells to determine the NF-κB signaling proteins associated with DUSP5. As shown in Fig. 5a and Supplementary Figure 3, IκBα, IKKα/β, and TAK1 were detected in immunoprecipitated DUSP5 complexes from RAW 264.7 cell lysates but not in immunoprecipitated pre-immune IgG complexes. We also confirmed that DUSP5 associates with TAK1, IKKα/β, and IκBα, in DUSP5 WT MEFs whereas no interaction was detected in DUSP5 KO cells (Supplementary Figure 4). These results imply that the NF-κB signaling pathway is regulated by the formation of a molecular complex between DUSP5 and NF-κB signal transduction molecules, including IκBα, IKKα/β, and TAK1.
However, the data from the co-immunoprecipitation between DUSP5 and endogenous NF-κB signaling molecules do not explain the fact that DUSP5 is not involved in the regulation of TAK1 phosphorylation even though  The phosphorylation levels of ERK1/2 were normalized to the corresponding total ERK1/2 levels, whereas those of p-IκBα and total IκBα were normalized to tubulin levels; all data are presented as fold increases. Similar results were obtained in three independent experiments. (b) DUSP5 WT-or C263Stransfected RAW 264.7 cells were stimulated with LPS (1 μg/ml) for 15 min. Immunoblots were probed with the indicated antibodies. The levels of p-IκBα and total IκBα were normalized to tubulin expression levels and presented as fold increases. The levels of p-IKKα/β and p-TAK1 were normalized to the expression levels of IKKα/β and TAK1. Similar results were obtained in three independent experiments. (c) DUSP5 WT and KO MEF cells were treated with TNF-α for 10 min and total cell lysates were then obtained. The same quantity of total proteins was subjected to immunoblotting analysis with appropriate antibodies. The levels of p-IκBα and total IκBα were normalized to tubulin expression levels. The levels of p-IKKα/β and p-TAK1 were normalized to the expression levels of IKKα/β and TAK1. The relative levels of p-IκBα, total IκBα, p-IKKα/β, and p-TAK1 were presented as fold increases. Similar results were obtained in three independent experiments. (d) RAW 264.7 cells were left untreated or stimulated with 1 μg/ml LPS for 2 h. Harvested cells were lysed (Total) or fractionated into cytoplasmic (C) and nuclear (N) fractions. Each fraction (30 μg for total cell lysate, 40 μg for cytoplasmic or nuclear fraction) was immunoblotted using specific antibodies. Anti-GAPDH and anti-Lamin B1 antibodies were used to verify the efficient fractionation of cytoplasmic and nuclear proteins, respectively. Similar results were obtained in three independent experiments. DUSP5 and TAK1 associate in cells. We, therefore, carried out in vitro binding assays to identify the direct interacting partners of DUSP5. FLAG-IκBα, -IKKα, -IKKβ, or -TAK1 proteins were purified from transfected cells by extensive washing after binding of FLAG-fused proteins to anti-FLAG M2 agarose and then incubated with purified recombinant GST-DUSP5. As shown in Fig. 5b, FLAG-IKKα, FLAG-IKKβ, and FLAG-TAK1 proteins interacted with GST-DUSP5 in vitro. However, GST-DUSP5 failed to interact with FLAG-IκBα. These results indicate that DUSP5-mediated dephosphorylation of p-IκBα is not due to a direct interaction between DUSP5 and IκBα.
These results led to a hypothesis that DUSP5-mediated regulation of NF-κB signal transduction might be conducted by the physical intervention of DUSP5 between IKKα/β and TAK1, and thereby inhibition of IKKα/β phosphorylation. We therefore investigated whether DUSP5 inhibits IKK binding to TAK1 in a dose-dependent manner in cells. Total cell lysates from cells transfected with HA-IKKβ, FLAG-TAK1, and increasing amounts of FLAG-DUSP5 WT or C263S expression plasmids, were subjected to immunoprecipitation using anti-HA antibodies. After removal of unbound proteins, the levels of DUSP5 and TAK1 bound to IKKβ were detected by immunoblotting analysis. With increasing DUSP5 WT or C263S, the levels of TAK1 bound to IKKβ were gradually reduced in a dose-dependent manner (Fig. 5c). Furthermore, DUSP5 WT failed to directly dephosphorylate p-IκBα and p-IKKα/β in vitro (Supplementary figure 5). These results indicate that DUSP5 acts as a competitor of TAK1 for binding to IKKβ and that its phosphatase activity is not necessary for this competition.

Discussion
DUSP5 is a potent pro-inflammatory regulator induced by IL-2, IL-7, and IL-15, and inhibits IL-2-induced ERK1/2 activation 33,34 . In this report, we showed that DUSP5 was transiently induced during LPS-mediated inflammatory responses in RAW 264.7 macrophages. In addition, transient DUSP5 overexpression suppressed TNF-α and IL-6 production through inactivation of both ERK1/2 and NF-κB pathways. Both the MAPK (ERK, JNK, and p38) and NF-κB signaling pathways are reportedly activated in RAW 264.7 macrophages upon exposure to LPS [35][36][37] . It has also been reported that DUSP5 expression is regulated mainly at the transcriptional level by the transcription factor Elk-1 38 , and that Elk-1 is phosphorylated and thus activated by ERK 39 , suggesting that LPS induces ERK-mediated Elk-1 activation and thus DUSP5 activity. Furthermore, sustained inflammation caused by NF-κB activation induced DUSP5 expression in irradiated human arteries 21 . These results imply that the transient induction of DUSP5 is involved in negative feedback regulation during the LPS-induced inflammatory response via inactivation of both ERK1/2 and NF-κB pathways. Since DUSP5 acts as a negative regulator of both ERK1/2 and NF-κB signal transduction in macrophages, the role of DUSP5 on signal transduction in the regulation of inflammatory responses may be more important than that of any other phosphatase.
That DUSP5 regulates NF-κB signal transduction in macrophages is a novel finding. In a previous genome-wide study, it was shown that knockdown of DUSP5 increased NF-κB activity, but the mechanism underlying the finding was not investigated 40 . DUSP5 failed to dephosphorylate p-IκB and several NF-κB-regulating phosphatases are known to act on NF-κB or other upstream kinases such as IKK or TRAF2 but not on IκB 41 , suggesting that DUSP5 might target upstream kinases. Interestingly, previous reports showed that DUSP5 is localized in the nucleus and regulates nuclear ERK activity 22,42 , but NF-κB upstream kinases are localized in the cytoplasm, which causes a subcellular localization conflict. However, our data and another report 43 show that DUSP5 is localized in both the cytoplasmic and nuclear fractions, suggesting the possibility of a physical association between DUSP5 and the NF-κB upstream kinases.
NF-κB activity might be regulated through the action of DUSP5 as a scaffold since DUSP5 interacts with both TAK1 and IKKs. In addition, DUSP proteins that act as scaffold proteins do not require their phosphatase activities 44,45 , which is the characteristic of DUSP5 for the regulation of NF-κB signaling. If DUSP5 acts as a scaffold in NF-κB signaling, DUSP5 should facilitate the association between TAK1 and IKKs. However, as shown in Fig. 5c, increase in DUSP5 concentration resulted in decreased association between the two proteins in a dose-dependent manner regardless of DUSP5 phosphatase activity, indicating that DUSP5 is not a scaffold protein.
In vitro phosphatase assays. For in vitro phosphatase assays, RAW 264.7 cells were treated with or without LPS and then harvested in PTP lysis buffer (1% IGEPAL CA-630, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM PMSF, and 1 μg/ml aprotinin) for 30 min at 4 °C. Cleared cell lysates from centrifugation were immunoprecipitated with rabbit anti-DUSP5 or anti-IgG antibodies (Santa Cruz) for 3 h at 4 °C followed by incubation with protein A/G agarose for 1 h at 4 °C using rotation device. After incubation, immunoprecipitates were washed three times with PTP lysis buffer and its phosphatase activities were measured using the substrate 3-O-methylfluorescein phosphate (OMFP; Sigma-Aldrich).
In vitro binding assay. HEK 293 cells were transfected with FLAG-tagged IκBα, IKKα, IKKβ, and TAK1 expression plasmid (1 μg) using the OmicsFect for 48 h. Total cell lysates were pulled down with anti-FLAG M2 agarose beads for 3 h and the pulled-down proteins were subjected to extensive washing to purify the FLAG-fusion proteins by excluding any bound proteins in the pulled-down complexes. To determine whether DUSP5 directly bind to IκBα, IKKα, IKKβ, or TAK1, each anti-FLAG bead-bound protein was mixed with GST-DUSP5 WT (2 μg) in 1 ml of PTP reaction buffer (100 mM Tris-HCl (pH 7.5), 40 mM NaCl, and 1 mM DTT) and incubated for 3 h at 4 °C. After incubation, the beads were washed 5 times with binding buffer, 1 X sample buffer was then added and boiled for 5 min at 100 °C. The samples were subjected to immunoblotting analyses using appropriate antibodies.
Enzyme linked immunosorbent assay (ELISA) of TNF-α and IL-6. TNF-α and IL-6 protein concentrations were determined by sandwich ELISA using antibodies and standards obtained from eBiosciences (San Diego, CA) according to manufacturer's instructions. Assays were performed on neat and diluted samples in triple on 96-well plates. Absorbance was measured by a microplate reader at 450 nm and concentrations were determined by comparison to a standard curve. All experiments were repeated at least three times. Immunoblotting analysis. Immunoblotting was performed with a SDS-PAGE Electrophoresis System as described previously 14 . Briefly, samples were run on SDS-10% polyacrylamide gels and transferred to nitrocellulose membrane. The membranes were blocked in 5% nonfat skim milk and incubated with an appropriate antibody, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. The immunoreactive bands were visualized using an ECL system (Pierce, Rockford, IL) and a cooled charge-device camera system (AE-9150, ATTO Technology, Tokyo, Japan). The intensity of the immunoreactive bands was quantified using LabWorks Analysis software (UVP, LLC, Upland, CA).

Reverse transcription-polymerase chain reaction (RT-PCR). Total RNAs were prepared from cells by
Accuzol (Bioneer Corporation, Daejon, Korea) and RT was performed using Omniscript RT Kit (Qiagen, Hilden, Germany). PCR for mouse PTPs was carried out using the primers listed in Table 1.
Quantitative Real-time PCR (qRT-PCR). Total RNAs prepared from RAW 264.7 cells using Accuzol (Bioneer Corporation) were reverse transcribed into cDNA and then qPCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). The following primer sets were used: DUSP5 mRNA (forward,