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  • 99
    Millipore sb203580
    LDH release after OGD injury in rat cortical astrocytes. Cells were subjected to 6 h of OGD, followed by 24 h of reoxygenation. Cell death was assessed by LDH release. <t>SB203580</t> (10 μM) significantly reduced OGD-induced
    Sb203580, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam sb203580
    Inhibition of p38 weakens heat stress-induced phosphorylation of MK5 and MK2. F98 cells underwent pretreatment with or without the indicated inhibitors for 30 min prior to exposure to heat stress or control heat treatment. After the cells received further incubation for 12 h at 37°C, antibodies specific for p-MK2, p-MK5, MK2, MK5 were used to assess the protein levels in the cell lysates. HS, heat stress; p-, phosphorylated; t-, total; MK, mitogen-activated protein kinase-activated protein kinase; <t>SB203580,</t> p38 inhibitor; PD98059, ERK inhibitor; SP600125, JNK inhibitor.
    Sb203580, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris sb203580
    Effect of 1α,25VitD3 on the expression of phospho-p38 MAPK in the peripheral blood neutrophil of the AECOPD patients. Neutrophils (1×10 6 ) were pretreated with <t>SB203580</t> (2.5 μM×10 –5 ) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10 -7 M) for 24 h. Western blot was used to analyze the phospho-p38 MAPK and p38 MAPK levels. The experiments shown are representative of at least 3 experiments. * p
    Sb203580, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb203580/product/Tocris
    Average 99 stars, based on 878 article reviews
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    86
    LKT Laboratories sb203580
    The role of p38 in mediating the pro-inflammatory effect of OE. (A) Increased p38 phosphorylation in OE-treated BEAS-2B cells. Cells were exposed to OE for 2 and 4 h. Total p38 was used as internal control. (B) Effect of p38 inhibitor on IL-6 production by OE-treated cells. (C). Effect of p38 inhibitor on OE-induced IL-8 production. BEAS-2B cells were pre-incubated with p38 inhibitor <t>SB203580</t> for 2 h prior to being exposed to OE for 16 h. OE50: OE at 50 μg/ml. Values represent means ± SEM, n = 3, * p
    Sb203580, supplied by LKT Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore inhibitors sb203580
    p38 induces E2F1 expression via MK2 upon epirubicin treatment A. <t>SB203580-treatment</t> induces JNK activation and downregulation of E2F1 and FOXM1 expression in response to doxorudicin . MCF-7 cells were either pretreated with 20 μM SB203580 or untreated and 1 h later exposed to 1 μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-p38 was immunoblotted as a loading control. B. Knockdown of p38 enhances the intensity and duration of JNK activation . U2OS cells were transfected with nontargeting siRNA (NS) or siRNAs specific to p38α and p38β. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control. C. MK2 mediates the p38-dependent repression of JNK activity . U2OS cells were transfected with either non-targeting siRNA (NS), Chk2 siRNA or MK2 siRNA. 48h later cells were treated with 1μM epirubicin and harvested at the indicated times. Whole cell lysates were prepared and equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. D. p38-dependent repression of JNK activity is mediated by MK2 and MK3 . Wild-type or MK2 -/- MK3 -/- MEFs were treated with 1μ epirubicin for 0, 4 or 8 h. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control.
    Inhibitors Sb203580, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    LDH release after OGD injury in rat cortical astrocytes. Cells were subjected to 6 h of OGD, followed by 24 h of reoxygenation. Cell death was assessed by LDH release. SB203580 (10 μM) significantly reduced OGD-induced

    Journal: Journal of Neurotrauma

    Article Title: Involvement of Mitogen-Activated Protein Kinase Pathways in Expression of the Water Channel Protein Aquaporin-4 after Ischemia in Rat Cortical Astrocytes

    doi: 10.1089/neu.2012.2430

    Figure Lengend Snippet: LDH release after OGD injury in rat cortical astrocytes. Cells were subjected to 6 h of OGD, followed by 24 h of reoxygenation. Cell death was assessed by LDH release. SB203580 (10 μM) significantly reduced OGD-induced

    Article Snippet: Thirty minutes before the ischemic insult, a total of 10 μL of 100 μM of SB203580 (100 μM dissolved in 1% dimethyl sulfoxide), a specific p38 inhibitor (Calbiochem), or vehicle (1% dimethyl sulfoxide), were injected stereotaxically into the right lateral cerebroventricle (coordinates with reference to the bregma: 1.4 mm lateral, 0.8 mm posterior, 3.6 mm deep) using a 26-G Hamilton microsyringe (80330; Hamilton Company, Reno, NV).

    Techniques:

    Infarct volume and edema volume 3 days after transient focal cerebral ischemia in vehicle-treated and SB203580-treated rats ( n =6 each; * p

    Journal: Journal of Neurotrauma

    Article Title: Involvement of Mitogen-Activated Protein Kinase Pathways in Expression of the Water Channel Protein Aquaporin-4 after Ischemia in Rat Cortical Astrocytes

    doi: 10.1089/neu.2012.2430

    Figure Lengend Snippet: Infarct volume and edema volume 3 days after transient focal cerebral ischemia in vehicle-treated and SB203580-treated rats ( n =6 each; * p

    Article Snippet: Thirty minutes before the ischemic insult, a total of 10 μL of 100 μM of SB203580 (100 μM dissolved in 1% dimethyl sulfoxide), a specific p38 inhibitor (Calbiochem), or vehicle (1% dimethyl sulfoxide), were injected stereotaxically into the right lateral cerebroventricle (coordinates with reference to the bregma: 1.4 mm lateral, 0.8 mm posterior, 3.6 mm deep) using a 26-G Hamilton microsyringe (80330; Hamilton Company, Reno, NV).

    Techniques:

    The cells were treated with 10 μM or 1 μM of PD98059, SP600125, or SB203580, before OGD. Sixteen hours after reoxygenation, the AQP4 level was determined by Western blot analysis. Ten micromoles of SB203580 or SP600125

    Journal: Journal of Neurotrauma

    Article Title: Involvement of Mitogen-Activated Protein Kinase Pathways in Expression of the Water Channel Protein Aquaporin-4 after Ischemia in Rat Cortical Astrocytes

    doi: 10.1089/neu.2012.2430

    Figure Lengend Snippet: The cells were treated with 10 μM or 1 μM of PD98059, SP600125, or SB203580, before OGD. Sixteen hours after reoxygenation, the AQP4 level was determined by Western blot analysis. Ten micromoles of SB203580 or SP600125

    Article Snippet: Thirty minutes before the ischemic insult, a total of 10 μL of 100 μM of SB203580 (100 μM dissolved in 1% dimethyl sulfoxide), a specific p38 inhibitor (Calbiochem), or vehicle (1% dimethyl sulfoxide), were injected stereotaxically into the right lateral cerebroventricle (coordinates with reference to the bregma: 1.4 mm lateral, 0.8 mm posterior, 3.6 mm deep) using a 26-G Hamilton microsyringe (80330; Hamilton Company, Reno, NV).

    Techniques: Western Blot

    ( A ) Suppression of p38 MAPK activity by administration of SB203580. p38 MAPK activity was examined 1 and 3 days after reperfusion with or without SB203580 administration. SB203580 was administered 30 min before ischemic insult, and phosphorylation

    Journal: Journal of Neurotrauma

    Article Title: Involvement of Mitogen-Activated Protein Kinase Pathways in Expression of the Water Channel Protein Aquaporin-4 after Ischemia in Rat Cortical Astrocytes

    doi: 10.1089/neu.2012.2430

    Figure Lengend Snippet: ( A ) Suppression of p38 MAPK activity by administration of SB203580. p38 MAPK activity was examined 1 and 3 days after reperfusion with or without SB203580 administration. SB203580 was administered 30 min before ischemic insult, and phosphorylation

    Article Snippet: Thirty minutes before the ischemic insult, a total of 10 μL of 100 μM of SB203580 (100 μM dissolved in 1% dimethyl sulfoxide), a specific p38 inhibitor (Calbiochem), or vehicle (1% dimethyl sulfoxide), were injected stereotaxically into the right lateral cerebroventricle (coordinates with reference to the bregma: 1.4 mm lateral, 0.8 mm posterior, 3.6 mm deep) using a 26-G Hamilton microsyringe (80330; Hamilton Company, Reno, NV).

    Techniques: Activity Assay

    Inhibition of p38 weakens heat stress-induced phosphorylation of MK5 and MK2. F98 cells underwent pretreatment with or without the indicated inhibitors for 30 min prior to exposure to heat stress or control heat treatment. After the cells received further incubation for 12 h at 37°C, antibodies specific for p-MK2, p-MK5, MK2, MK5 were used to assess the protein levels in the cell lysates. HS, heat stress; p-, phosphorylated; t-, total; MK, mitogen-activated protein kinase-activated protein kinase; SB203580, p38 inhibitor; PD98059, ERK inhibitor; SP600125, JNK inhibitor.

    Journal: Oncology Letters

    Article Title: p38 MAPK-MK2 pathway regulates the heat-stress-induced accumulation of reactive oxygen species that mediates apoptotic cell death in glial cells

    doi: 10.3892/ol.2017.7360

    Figure Lengend Snippet: Inhibition of p38 weakens heat stress-induced phosphorylation of MK5 and MK2. F98 cells underwent pretreatment with or without the indicated inhibitors for 30 min prior to exposure to heat stress or control heat treatment. After the cells received further incubation for 12 h at 37°C, antibodies specific for p-MK2, p-MK5, MK2, MK5 were used to assess the protein levels in the cell lysates. HS, heat stress; p-, phosphorylated; t-, total; MK, mitogen-activated protein kinase-activated protein kinase; SB203580, p38 inhibitor; PD98059, ERK inhibitor; SP600125, JNK inhibitor.

    Article Snippet: Cells were pretreated with CMPD-1 (10 µM; cat. no. 263847-55-8; Tocris Bioscience, Bristol, UK), MnTBAP (10 µM; cat. no. ab141496; Abcam, Cambridge, MA, USA), SP600125 (10 µM; cat. no. ab120065; Abcam), SB203580 (5 µM; cat. no. ab120162; Abcam), PD98059 (10 µM; cat. no. ab120234; Abcam), SB202474 (10 µM; cat. no. IMA1013; Jingke Science and Technology Co., Ltd., Shanghai, China), the specific inhibitors of MK2, ROS, JNK, p38, ERK and the negative control, respectively, at 37°C for 30 min.

    Techniques: Inhibition, Incubation

    SFN inhibitory effect on p38 and JNK impacts on S. aureus -mediated expression of proinflammatory genes and S. aureus survival in macrophages. (A-C) THP-1-derived macrophages were pretreated with p38 inhibitor SB or JNK inhibitor SP for 1 h prior to 10 µ M SFN or DMSO treatment. After additional 3 h incubation, cells were infected with S. aureus . Total RNA was extracted 3 h post-infection and (A) human IL-1β, (B) IL-6 and (C) TNF-α mRNA expression levels were determined by RT-qPCR (n=4). (D-F) Nrf2 +/− BMDMs were pretreated with SB203580 and SP600125 prior to 10 µ M SFN or DMSO treatment and S. aureus infection. (D) Mouse IL-1β, (E) IL-6 and (F) TNF-α mRNA expression levels were quantified by RT-qPCR (n=5 mice). (G) Intracellular survival of S. aureus was assessed in THP-1-derived macrophages pretreated with SB and SP prior to 10 µ M SFN or DMSO treatment and S. aureus infection. CFU counts were determined 24 h after infection (n=4). * P

    Journal: International Journal of Molecular Medicine

    Article Title: Sulforaphane reduces intracellular survival of Staphylococcus aureus in macrophages through inhibition of JNK and p38 MAPK-induced inflammation

    doi: 10.3892/ijmm.2020.4563

    Figure Lengend Snippet: SFN inhibitory effect on p38 and JNK impacts on S. aureus -mediated expression of proinflammatory genes and S. aureus survival in macrophages. (A-C) THP-1-derived macrophages were pretreated with p38 inhibitor SB or JNK inhibitor SP for 1 h prior to 10 µ M SFN or DMSO treatment. After additional 3 h incubation, cells were infected with S. aureus . Total RNA was extracted 3 h post-infection and (A) human IL-1β, (B) IL-6 and (C) TNF-α mRNA expression levels were determined by RT-qPCR (n=4). (D-F) Nrf2 +/− BMDMs were pretreated with SB203580 and SP600125 prior to 10 µ M SFN or DMSO treatment and S. aureus infection. (D) Mouse IL-1β, (E) IL-6 and (F) TNF-α mRNA expression levels were quantified by RT-qPCR (n=5 mice). (G) Intracellular survival of S. aureus was assessed in THP-1-derived macrophages pretreated with SB and SP prior to 10 µ M SFN or DMSO treatment and S. aureus infection. CFU counts were determined 24 h after infection (n=4). * P

    Article Snippet: The Annexin V-fluorescein isothiocyanate apoptosis reagent, and the inhibitors SB203580 and SP600125 were obtained from Abcam.

    Techniques: Expressing, Derivative Assay, Incubation, Infection, Quantitative RT-PCR, Mouse Assay

    Effect of 1α,25VitD3 on the expression of phospho-p38 MAPK in the peripheral blood neutrophil of the AECOPD patients. Neutrophils (1×10 6 ) were pretreated with SB203580 (2.5 μM×10 –5 ) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10 -7 M) for 24 h. Western blot was used to analyze the phospho-p38 MAPK and p38 MAPK levels. The experiments shown are representative of at least 3 experiments. * p

    Journal: PLoS ONE

    Article Title: 1α,25-Dihydroxyvitamin D3 Induces Neutrophil Apoptosis through the p38 MAPK Signaling Pathway in Chronic Obstructive Pulmonary Disease Patients

    doi: 10.1371/journal.pone.0120515

    Figure Lengend Snippet: Effect of 1α,25VitD3 on the expression of phospho-p38 MAPK in the peripheral blood neutrophil of the AECOPD patients. Neutrophils (1×10 6 ) were pretreated with SB203580 (2.5 μM×10 –5 ) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10 -7 M) for 24 h. Western blot was used to analyze the phospho-p38 MAPK and p38 MAPK levels. The experiments shown are representative of at least 3 experiments. * p

    Article Snippet: Neutrophils from the COPD patients were divided into 4 groups: 1) the COPD control group (Group B); 2) the SB203580(+) + 1α,25VitD3 COPD group (Group C) (Tocris Bioscience, Ellisville, MO), which was pretreated with 2.5 μM × 10–5 SB203580 for 0.5 h, followed by incubation with 1α,25VitD3 (6 × 10–7 M) (Sigma-Aldrich); 3) the 1α,25VitD3 group (Group D), which was treated with 6 × 10–7 M 1α,25VitD3; 4) the COPD SB203580(+) group (Group E), which was pretreated with 2.5 μM × 10–5 SB203580 as described for group 2.

    Techniques: Expressing, Incubation, Western Blot

    1α,25VitD3 increased peripheral blood neutrophil apoptosis in the AECOPD patients. Neutrophils (1 ×10 6 ) were pretreated with SB203580 (2.5 μM × 10 –5 ) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10 –7 M) for 24 h. Neutrophil apoptosis was analyzed with flow cytometry (a). The right lower quadrant (RLQ) contains early apoptotic neutrophils with positive Annexin V-PE binding and negative 7-AAD binding (Annexin V-PE [z], 7-AAD [–]). The vital neutrophils in the lower left quadrant (LLQ) are Annexin V-PE (-),7-AAD (-). The upper right quadrant (RUQ) contains late apoptotic and necrotic neutrophils (Annexin V-PE [z], 7-AAD [z]). The amount of neutrophil apoptosis in the RUD and RLD quadrants are shown in (b), expressed as the % of the total cell count (10,000).The experiments shown are representative of at least 3 experiments. The data are presented as the means ± sd, n = 36,* p

    Journal: PLoS ONE

    Article Title: 1α,25-Dihydroxyvitamin D3 Induces Neutrophil Apoptosis through the p38 MAPK Signaling Pathway in Chronic Obstructive Pulmonary Disease Patients

    doi: 10.1371/journal.pone.0120515

    Figure Lengend Snippet: 1α,25VitD3 increased peripheral blood neutrophil apoptosis in the AECOPD patients. Neutrophils (1 ×10 6 ) were pretreated with SB203580 (2.5 μM × 10 –5 ) for 0.5 h, followed by incubation with 1α,25VitD3 (6 ×10 –7 M) for 24 h. Neutrophil apoptosis was analyzed with flow cytometry (a). The right lower quadrant (RLQ) contains early apoptotic neutrophils with positive Annexin V-PE binding and negative 7-AAD binding (Annexin V-PE [z], 7-AAD [–]). The vital neutrophils in the lower left quadrant (LLQ) are Annexin V-PE (-),7-AAD (-). The upper right quadrant (RUQ) contains late apoptotic and necrotic neutrophils (Annexin V-PE [z], 7-AAD [z]). The amount of neutrophil apoptosis in the RUD and RLD quadrants are shown in (b), expressed as the % of the total cell count (10,000).The experiments shown are representative of at least 3 experiments. The data are presented as the means ± sd, n = 36,* p

    Article Snippet: Neutrophils from the COPD patients were divided into 4 groups: 1) the COPD control group (Group B); 2) the SB203580(+) + 1α,25VitD3 COPD group (Group C) (Tocris Bioscience, Ellisville, MO), which was pretreated with 2.5 μM × 10–5 SB203580 for 0.5 h, followed by incubation with 1α,25VitD3 (6 × 10–7 M) (Sigma-Aldrich); 3) the 1α,25VitD3 group (Group D), which was treated with 6 × 10–7 M 1α,25VitD3; 4) the COPD SB203580(+) group (Group E), which was pretreated with 2.5 μM × 10–5 SB203580 as described for group 2.

    Techniques: Incubation, Flow Cytometry, Cytometry, Binding Assay, Cell Counting

    p38 MAPK controls IL-17 production at the posttranscriptional level. (A) FACS-sorted CD4 T cells from WT B6 mice were differentiated into Th17 cells in the absence or presence of SB203580 (5μM) for the indicated periods of time. The phosphorylation of STAT3 at Ser727 (P-Ser) or at Tyr705 (P-Tyr) was examined by Western blot analysis. Total STAT3 (Total) is also shown. FACS-sorted CD4 T cells from WT mice were activated under Th17 conditions in the absence (control) or presence of SB203580 for 48h. Relative Rorc (B) and Il17 (C) mRNA levels were examined by quantitative real-time PCR using β2-microglobulin as the endogenous control. (D) Relative Il17 mRNA levels in FACS-sorted CD4 T cells from WT B6 and MKK3 −/− MKK6 +/− mice activated under Th17 conditions for 48h. (E) Intracellular staining for IL-17 and IFNγ in WT total CD4 T cells differentiated into Th17 cells in the absence (control) or presence of SB203580 for 72 hours.

    Journal: Blood

    Article Title: Activation of p38 MAPK in CD4 T cells controls IL-17 production and autoimmune encephalomyelitis

    doi: 10.1182/blood-2011-02-336552

    Figure Lengend Snippet: p38 MAPK controls IL-17 production at the posttranscriptional level. (A) FACS-sorted CD4 T cells from WT B6 mice were differentiated into Th17 cells in the absence or presence of SB203580 (5μM) for the indicated periods of time. The phosphorylation of STAT3 at Ser727 (P-Ser) or at Tyr705 (P-Tyr) was examined by Western blot analysis. Total STAT3 (Total) is also shown. FACS-sorted CD4 T cells from WT mice were activated under Th17 conditions in the absence (control) or presence of SB203580 for 48h. Relative Rorc (B) and Il17 (C) mRNA levels were examined by quantitative real-time PCR using β2-microglobulin as the endogenous control. (D) Relative Il17 mRNA levels in FACS-sorted CD4 T cells from WT B6 and MKK3 −/− MKK6 +/− mice activated under Th17 conditions for 48h. (E) Intracellular staining for IL-17 and IFNγ in WT total CD4 T cells differentiated into Th17 cells in the absence (control) or presence of SB203580 for 72 hours.

    Article Snippet: Mice received 5 mg/kg/d SB203580 dihydrochloride (Tocris) by IP injection in a total volume of 200 μL or an equal volume of carrier everyday from the day of immunization.

    Techniques: FACS, Mouse Assay, Western Blot, Real-time Polymerase Chain Reaction, Staining

    p38 MAPK controls IL-17 production by Th17 cells in vivo. (A) Mononuclear cells were isolated from the CNS of 2× MOG 35-55 -CFA immunized mice on day 35 after immunization, stimulated with PMA/ionomycin for 4 hours in the presence of brefeldin A, stained, and analyzed by flow cytometry. (Left panel) Cells were gated on CD45 and the frequency of CD4 + TCRβ + cells was determined. (Right panels) Cells were gated on CD4 and TCRβ and the frequency of IL-17 + and IFNγ + cells was determined. (B) Spleen and draining lymph node (DLN) cells from 2× MOG 35-55 -CFA immunized mice treated with either carrier (n = 7) or the SB203580 (n = 8) were harvested on day 21 after immunization and stimulated with MOG 35-55 (50 μg/mL) for 72 hours and IL-17 and IFNγ in the supernatants was quantified by ELISA. (C) Spleen and DLN cells from 2× MOG 35-55 -CFA immunized mice (n = 10) were harvested on day 10 after immunization and stimulated with MOG 35-55 in the presence or absence of SB203580 (SB; 5μM) for 72 hours. IL-17 production was quantified by ELISA. (D) Cells obtained as in panel C were stimulated with the indicated amounts of MOG 35-55 in the presence or the absence of SB203580 and proliferation was assessed by [ 3 H]-thymidine incorporation. The significance of differences observed in panels A through C was determined using the Student t test (* ≤ 0.05; *** ≤ 0.001), and by 2-way ANOVA for panel D (SB, P = .31; MOG 35-55 , P = .003; and interaction, P = .95).

    Journal: Blood

    Article Title: Activation of p38 MAPK in CD4 T cells controls IL-17 production and autoimmune encephalomyelitis

    doi: 10.1182/blood-2011-02-336552

    Figure Lengend Snippet: p38 MAPK controls IL-17 production by Th17 cells in vivo. (A) Mononuclear cells were isolated from the CNS of 2× MOG 35-55 -CFA immunized mice on day 35 after immunization, stimulated with PMA/ionomycin for 4 hours in the presence of brefeldin A, stained, and analyzed by flow cytometry. (Left panel) Cells were gated on CD45 and the frequency of CD4 + TCRβ + cells was determined. (Right panels) Cells were gated on CD4 and TCRβ and the frequency of IL-17 + and IFNγ + cells was determined. (B) Spleen and draining lymph node (DLN) cells from 2× MOG 35-55 -CFA immunized mice treated with either carrier (n = 7) or the SB203580 (n = 8) were harvested on day 21 after immunization and stimulated with MOG 35-55 (50 μg/mL) for 72 hours and IL-17 and IFNγ in the supernatants was quantified by ELISA. (C) Spleen and DLN cells from 2× MOG 35-55 -CFA immunized mice (n = 10) were harvested on day 10 after immunization and stimulated with MOG 35-55 in the presence or absence of SB203580 (SB; 5μM) for 72 hours. IL-17 production was quantified by ELISA. (D) Cells obtained as in panel C were stimulated with the indicated amounts of MOG 35-55 in the presence or the absence of SB203580 and proliferation was assessed by [ 3 H]-thymidine incorporation. The significance of differences observed in panels A through C was determined using the Student t test (* ≤ 0.05; *** ≤ 0.001), and by 2-way ANOVA for panel D (SB, P = .31; MOG 35-55 , P = .003; and interaction, P = .95).

    Article Snippet: Mice received 5 mg/kg/d SB203580 dihydrochloride (Tocris) by IP injection in a total volume of 200 μL or an equal volume of carrier everyday from the day of immunization.

    Techniques: In Vivo, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    p38 MAPK regulates IL-17 production by in vitro–generated Th17 cells. FACS-sorted CD4 T cells from WT B6 mice in vitro differentiated into Th17 cells in the presence of different concentrations of SB203580 (A) or BIRB796 (B) for 72 hours. IL-17 production by Th17-polarized FACS-sorted CD4 T cells from WT B6 or MKK3 −/− MKK6 +/− mice (C) or total CD4 T cells from WT B10.BR and dn-p38-Tg (D) or MKK6 Tg (E) differentiated into Th17 cells. IL-17 levels in the supernatants were assessed by ELISA. The significance of differences observed in panels A and B was determined by linear regression analysis (A, P

    Journal: Blood

    Article Title: Activation of p38 MAPK in CD4 T cells controls IL-17 production and autoimmune encephalomyelitis

    doi: 10.1182/blood-2011-02-336552

    Figure Lengend Snippet: p38 MAPK regulates IL-17 production by in vitro–generated Th17 cells. FACS-sorted CD4 T cells from WT B6 mice in vitro differentiated into Th17 cells in the presence of different concentrations of SB203580 (A) or BIRB796 (B) for 72 hours. IL-17 production by Th17-polarized FACS-sorted CD4 T cells from WT B6 or MKK3 −/− MKK6 +/− mice (C) or total CD4 T cells from WT B10.BR and dn-p38-Tg (D) or MKK6 Tg (E) differentiated into Th17 cells. IL-17 levels in the supernatants were assessed by ELISA. The significance of differences observed in panels A and B was determined by linear regression analysis (A, P

    Article Snippet: Mice received 5 mg/kg/d SB203580 dihydrochloride (Tocris) by IP injection in a total volume of 200 μL or an equal volume of carrier everyday from the day of immunization.

    Techniques: In Vitro, Generated, FACS, Mouse Assay, Enzyme-linked Immunosorbent Assay

    In vivo inhibition of p38 MAPK prevents arginase-induced endothelial dysfunction and decreases arginase activity of diabetic MA. (A) Endothelium-dependent relaxations in response to ACh in mesenteric segments from DPBS-treated healthy (HS), SB203580-treated

    Journal: British Journal of Pharmacology

    Article Title: Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    doi: 10.1111/bph.13242

    Figure Lengend Snippet: In vivo inhibition of p38 MAPK prevents arginase-induced endothelial dysfunction and decreases arginase activity of diabetic MA. (A) Endothelium-dependent relaxations in response to ACh in mesenteric segments from DPBS-treated healthy (HS), SB203580-treated

    Article Snippet: The sources of the compounds used were as follows: ACh and SNP were obtained from Sigma Aldrich, U46619 and SB203580 hydrochloride from Tocris, Biosciences (Bristol, UK), and ABH from Enzo Clinical Labs (Farmingdale, NY, USA).

    Techniques: In Vivo, Inhibition, Activity Assay

    Effect of p38 MAPK inhibition on eNOS protein expression in mesenteric and coronary arteries. Western blot analysis of total eNOS and phospho-eNOS (Ser 1177 ) from coronary (A) and mesenteric (B) arteries of DPBS-treated healthy (HS), SB203580-treated healthy

    Journal: British Journal of Pharmacology

    Article Title: Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    doi: 10.1111/bph.13242

    Figure Lengend Snippet: Effect of p38 MAPK inhibition on eNOS protein expression in mesenteric and coronary arteries. Western blot analysis of total eNOS and phospho-eNOS (Ser 1177 ) from coronary (A) and mesenteric (B) arteries of DPBS-treated healthy (HS), SB203580-treated healthy

    Article Snippet: The sources of the compounds used were as follows: ACh and SNP were obtained from Sigma Aldrich, U46619 and SB203580 hydrochloride from Tocris, Biosciences (Bristol, UK), and ABH from Enzo Clinical Labs (Farmingdale, NY, USA).

    Techniques: Inhibition, Expressing, Western Blot

    p38 MAPK activation increases arginase II expression in MA. Western blot analysis of arginase II (A), arginase I (B) and ratio of phospho-p38 MAPK/total p38 MAPK (C) in MA of DPBS-treated healthy (HS), SB203580-treated healthy (HSB), DPBS-treated diabetic

    Journal: British Journal of Pharmacology

    Article Title: Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    doi: 10.1111/bph.13242

    Figure Lengend Snippet: p38 MAPK activation increases arginase II expression in MA. Western blot analysis of arginase II (A), arginase I (B) and ratio of phospho-p38 MAPK/total p38 MAPK (C) in MA of DPBS-treated healthy (HS), SB203580-treated healthy (HSB), DPBS-treated diabetic

    Article Snippet: The sources of the compounds used were as follows: ACh and SNP were obtained from Sigma Aldrich, U46619 and SB203580 hydrochloride from Tocris, Biosciences (Bristol, UK), and ABH from Enzo Clinical Labs (Farmingdale, NY, USA).

    Techniques: Activation Assay, Expressing, Western Blot

    Arginase and p38 MAPK inhibition improves relaxation in diabetic coronary arteries. (A, B) Endothelium-dependent relaxation in response to ACh from healthy and diabetic SCA (A) and LAD (B). (C, D) Effect of ABH (+ABH), SB203580 (+SB) and SB203580 plus

    Journal: British Journal of Pharmacology

    Article Title: Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    doi: 10.1111/bph.13242

    Figure Lengend Snippet: Arginase and p38 MAPK inhibition improves relaxation in diabetic coronary arteries. (A, B) Endothelium-dependent relaxation in response to ACh from healthy and diabetic SCA (A) and LAD (B). (C, D) Effect of ABH (+ABH), SB203580 (+SB) and SB203580 plus

    Article Snippet: The sources of the compounds used were as follows: ACh and SNP were obtained from Sigma Aldrich, U46619 and SB203580 hydrochloride from Tocris, Biosciences (Bristol, UK), and ABH from Enzo Clinical Labs (Farmingdale, NY, USA).

    Techniques: Inhibition

    p38 MAPK activation increases arginase I expression in diabetic coronary arteries. Western blot analysis of arginase I (A), arginase II (B) and ratio of phospho-p38 MAPK/total p38MAPK (C) in coronary arteries of DPBS-treated healthy (HS), SB203580-treated

    Journal: British Journal of Pharmacology

    Article Title: Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    doi: 10.1111/bph.13242

    Figure Lengend Snippet: p38 MAPK activation increases arginase I expression in diabetic coronary arteries. Western blot analysis of arginase I (A), arginase II (B) and ratio of phospho-p38 MAPK/total p38MAPK (C) in coronary arteries of DPBS-treated healthy (HS), SB203580-treated

    Article Snippet: The sources of the compounds used were as follows: ACh and SNP were obtained from Sigma Aldrich, U46619 and SB203580 hydrochloride from Tocris, Biosciences (Bristol, UK), and ABH from Enzo Clinical Labs (Farmingdale, NY, USA).

    Techniques: Activation Assay, Expressing, Western Blot

    Arginase and p38 MAPK inhibition improve endothelium-dependent relaxation in diabetic MA. (A) Endothelium-dependent relaxation in response to ACh in MA from healthy and diabetic rats. (B, C) Effects of ABH (+ABH), SB203580 (+SB) and SB+ABH on ACh-induced

    Journal: British Journal of Pharmacology

    Article Title: Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    doi: 10.1111/bph.13242

    Figure Lengend Snippet: Arginase and p38 MAPK inhibition improve endothelium-dependent relaxation in diabetic MA. (A) Endothelium-dependent relaxation in response to ACh in MA from healthy and diabetic rats. (B, C) Effects of ABH (+ABH), SB203580 (+SB) and SB+ABH on ACh-induced

    Article Snippet: The sources of the compounds used were as follows: ACh and SNP were obtained from Sigma Aldrich, U46619 and SB203580 hydrochloride from Tocris, Biosciences (Bristol, UK), and ABH from Enzo Clinical Labs (Farmingdale, NY, USA).

    Techniques: Inhibition

    Influence of kinase inhibitors on ppNOC and NOP mRNA expression. ppNOC and NOP expression in blood leukocytes cultured with PMA or without (control) and with or without pre-treatment with the specific kinase inhibitors PD98059 (PD, ERK inhibitor) 30 μM, SB203580 (SB, p38 inhibitor) 10 μM, SP600125 (SP, JNK inhibitor) 10 μM, Bay 11-7821 (Bay, NFκB inhibitor) 3 μM, or the combination of PD and SB. Box-and-whisker plots, medians with interquartile ranges, and 5 to 95 percentiles, n =10 for the PD+SB+PMA group, n =17 for all other groups. * P

    Journal: Molecular Pain

    Article Title: ERK and p38 contribute to the regulation of nociceptin and the nociceptin receptor in human peripheral blood leukocytes

    doi: 10.1177/1744806919828921

    Figure Lengend Snippet: Influence of kinase inhibitors on ppNOC and NOP mRNA expression. ppNOC and NOP expression in blood leukocytes cultured with PMA or without (control) and with or without pre-treatment with the specific kinase inhibitors PD98059 (PD, ERK inhibitor) 30 μM, SB203580 (SB, p38 inhibitor) 10 μM, SP600125 (SP, JNK inhibitor) 10 μM, Bay 11-7821 (Bay, NFκB inhibitor) 3 μM, or the combination of PD and SB. Box-and-whisker plots, medians with interquartile ranges, and 5 to 95 percentiles, n =10 for the PD+SB+PMA group, n =17 for all other groups. * P

    Article Snippet: Blood was pre-treated with PD98059 (PD) 30 μM, SP600125 (SP) 10 μM, SB203580 (SB) 10 μM, Bay 11-7871 (Bay) 3 μM, or the combination of PD and SB (all from Tocris Bioscience, Bristol, UK) for 1 h prior to culturing with or without PMA 10 ng/ml for 6 and 24 h. The concentrations of the inhibitors were based on doses used in previous studies., , A culture without any stimulus and one cultured only with PMA 10 ng/ml served as control group and reference group, respectively.

    Techniques: Expressing, Cell Culture, Whisker Assay

    The role of p38 in mediating the pro-inflammatory effect of OE. (A) Increased p38 phosphorylation in OE-treated BEAS-2B cells. Cells were exposed to OE for 2 and 4 h. Total p38 was used as internal control. (B) Effect of p38 inhibitor on IL-6 production by OE-treated cells. (C). Effect of p38 inhibitor on OE-induced IL-8 production. BEAS-2B cells were pre-incubated with p38 inhibitor SB203580 for 2 h prior to being exposed to OE for 16 h. OE50: OE at 50 μg/ml. Values represent means ± SEM, n = 3, * p

    Journal: Toxicology Reports

    Article Title: Emissions from commercial-grade charbroiling meat operations induce oxidative stress and inflammatory responses in human bronchial epithelial cells

    doi: 10.1016/j.toxrep.2014.09.015

    Figure Lengend Snippet: The role of p38 in mediating the pro-inflammatory effect of OE. (A) Increased p38 phosphorylation in OE-treated BEAS-2B cells. Cells were exposed to OE for 2 and 4 h. Total p38 was used as internal control. (B) Effect of p38 inhibitor on IL-6 production by OE-treated cells. (C). Effect of p38 inhibitor on OE-induced IL-8 production. BEAS-2B cells were pre-incubated with p38 inhibitor SB203580 for 2 h prior to being exposed to OE for 16 h. OE50: OE at 50 μg/ml. Values represent means ± SEM, n = 3, * p

    Article Snippet: R,S-Sulforaphane (SFN) and SB203580 were purchased from LKT Laboratories (St. Paul, MN) and EMD Millipore (Bedford, MA), respectively.

    Techniques: Incubation

    Up-regulation of COX-2 and PGE2 by OE. (A) Increased COX-2 protein in BEAS-2B cells treated with OE for 16 h. (B) Effects of NAC (1 mM), SFN (5 μM) and SB203580 (5 μM) on PGE2 release from BEAS-2B cells after16-h exposure to 50 μg/ml of OE. Values represent means ± SEM, n = 3. * p

    Journal: Toxicology Reports

    Article Title: Emissions from commercial-grade charbroiling meat operations induce oxidative stress and inflammatory responses in human bronchial epithelial cells

    doi: 10.1016/j.toxrep.2014.09.015

    Figure Lengend Snippet: Up-regulation of COX-2 and PGE2 by OE. (A) Increased COX-2 protein in BEAS-2B cells treated with OE for 16 h. (B) Effects of NAC (1 mM), SFN (5 μM) and SB203580 (5 μM) on PGE2 release from BEAS-2B cells after16-h exposure to 50 μg/ml of OE. Values represent means ± SEM, n = 3. * p

    Article Snippet: R,S-Sulforaphane (SFN) and SB203580 were purchased from LKT Laboratories (St. Paul, MN) and EMD Millipore (Bedford, MA), respectively.

    Techniques:

    p38 induces E2F1 expression via MK2 upon epirubicin treatment A. SB203580-treatment induces JNK activation and downregulation of E2F1 and FOXM1 expression in response to doxorudicin . MCF-7 cells were either pretreated with 20 μM SB203580 or untreated and 1 h later exposed to 1 μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-p38 was immunoblotted as a loading control. B. Knockdown of p38 enhances the intensity and duration of JNK activation . U2OS cells were transfected with nontargeting siRNA (NS) or siRNAs specific to p38α and p38β. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control. C. MK2 mediates the p38-dependent repression of JNK activity . U2OS cells were transfected with either non-targeting siRNA (NS), Chk2 siRNA or MK2 siRNA. 48h later cells were treated with 1μM epirubicin and harvested at the indicated times. Whole cell lysates were prepared and equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. D. p38-dependent repression of JNK activity is mediated by MK2 and MK3 . Wild-type or MK2 -/- MK3 -/- MEFs were treated with 1μ epirubicin for 0, 4 or 8 h. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control.

    Journal: Molecular cancer research : MCR

    Article Title: The p38 MAPK-MK2 axis regulates E2F1 and FOXM1 expression after epirubicin treatment

    doi: 10.1158/1541-7786.MCR-11-0559

    Figure Lengend Snippet: p38 induces E2F1 expression via MK2 upon epirubicin treatment A. SB203580-treatment induces JNK activation and downregulation of E2F1 and FOXM1 expression in response to doxorudicin . MCF-7 cells were either pretreated with 20 μM SB203580 or untreated and 1 h later exposed to 1 μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-p38 was immunoblotted as a loading control. B. Knockdown of p38 enhances the intensity and duration of JNK activation . U2OS cells were transfected with nontargeting siRNA (NS) or siRNAs specific to p38α and p38β. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control. C. MK2 mediates the p38-dependent repression of JNK activity . U2OS cells were transfected with either non-targeting siRNA (NS), Chk2 siRNA or MK2 siRNA. 48h later cells were treated with 1μM epirubicin and harvested at the indicated times. Whole cell lysates were prepared and equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. D. p38-dependent repression of JNK activity is mediated by MK2 and MK3 . Wild-type or MK2 -/- MK3 -/- MEFs were treated with 1μ epirubicin for 0, 4 or 8 h. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control.

    Article Snippet: The inhibitors SB203580 and SP600125 were acquired from Calbiochem (Merck), while SB202190 was from Sigma.

    Techniques: Expressing, Activation Assay, Western Blot, Transfection, Activity Assay

    p38 MAPK mediates E2F1 and FOXM1 induction in response to epirubicin A. Pretreatment with the p38 inhibitor SB203580 prevents epirubicin-induced E2F1 accumulation and its phosphorylation at Serine 364 . U2OS cells were either pretreated with 20 μM SB203580 or untreated and 1h later exposed to 1μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted from lysates. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. B. The p38 inhibitor SB203580 reduces the expression of epirubicin-induced E2F1 in a dose-dependent manner, accompanied by a corresponding decrease in FOXM1 protein levels . Total cell lysates derived from U2OS cells pretreated with increasing concentrations of SB203580 (0, 5, 10, 20 μM) followed by 6h exposure to 1 μM epirubicin were subjected to Western blot analysis. An untreated sample (C-) was loaded as a negative control. Anti-β-tubulin served as a loading control. C. siRNA-mediated depletion of p38 impairs E2F1 Ser-364 phosphorylation and FOXM1 upregulation induced by epirubicin . U2OS cells were transfected with nontargeting siRNA (NS) or a mixture of siRNAs specific to p38α and p38β. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control.

    Journal: Molecular cancer research : MCR

    Article Title: The p38 MAPK-MK2 axis regulates E2F1 and FOXM1 expression after epirubicin treatment

    doi: 10.1158/1541-7786.MCR-11-0559

    Figure Lengend Snippet: p38 MAPK mediates E2F1 and FOXM1 induction in response to epirubicin A. Pretreatment with the p38 inhibitor SB203580 prevents epirubicin-induced E2F1 accumulation and its phosphorylation at Serine 364 . U2OS cells were either pretreated with 20 μM SB203580 or untreated and 1h later exposed to 1μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted from lysates. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. B. The p38 inhibitor SB203580 reduces the expression of epirubicin-induced E2F1 in a dose-dependent manner, accompanied by a corresponding decrease in FOXM1 protein levels . Total cell lysates derived from U2OS cells pretreated with increasing concentrations of SB203580 (0, 5, 10, 20 μM) followed by 6h exposure to 1 μM epirubicin were subjected to Western blot analysis. An untreated sample (C-) was loaded as a negative control. Anti-β-tubulin served as a loading control. C. siRNA-mediated depletion of p38 impairs E2F1 Ser-364 phosphorylation and FOXM1 upregulation induced by epirubicin . U2OS cells were transfected with nontargeting siRNA (NS) or a mixture of siRNAs specific to p38α and p38β. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control.

    Article Snippet: The inhibitors SB203580 and SP600125 were acquired from Calbiochem (Merck), while SB202190 was from Sigma.

    Techniques: Western Blot, Expressing, Derivative Assay, Negative Control, Transfection

    FOXM1 phosphorylation is responsible for the induction of E2F1 protein expression in response to epirubicin A. Neither Ser-364 nor Ser-31 is solely responsible for the induction of E2F1 protein expression in response to epirubicin . U2OS cells were transfected with either the empty pCMV plasmid, pCMV-E2F1-132E, wild-type pCMV-HA-E2F1 plasmid, or an pCMV-HA-E2F1 mutant plasmid bearing one of the following point mutations: S31A, S31D, S364A or S364D. Thirty–six h later transfected cells were either untreated or exposed to epirubicin for 6 h and harvested. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the antibodies indicated. B. E2F1 phosphorylation mutants have depleted transactivation activity . Four thousand cells were seeded in each well of a 96-well plate, allowed to attach overnight and transfected with 20 ng of the indicated expression plasmids together with 5ng Renilla reporter and 20 ng of the luciferase reporter gene driven by the E2F1 promoter. Twenty h later, 6 replicates of each condition were left untreated while 6 others were treated with 1 μM epirubicin for 3 h prior to measurement of luciferase activity. Graphs represent mean (± SD) luciferase activity corrected for Renilla luciferase activity. Values were normalised to that of the empty vector in untreated cells. C. S364-phosphorylation contributes to E2F1 activation . 4000 cells were seeded in 96-well plates, allowed to attach overnight and transfected with either empty vector, pCMV-E2F1-132E or increasing concentrations (0, 10, 20 or 30 ng) of wild-type HA-E2F1 or the E2F1-S364A point mutant together with 5 ng Renilla reporter and 20 ng of the luciferase reporter gene driven by the E2F1 promoter. 6 replicates of each combination were measured. Graphs represent mean (± SD) luciferase activity in corrected for Renilla luciferase activity. Values were normalised to that of empty vector (-). D. MK2 and p38 contribute to FOXM1 transactivation by E2F1 . U2OS cells were transfected with 20 ng of either empty vector, pCMV-E2F1-132E or wild-type HA-E2F1 together with 5 ng Renilla reporter and 20 ng of the luciferase reporter gene driven by the FOXM1 ) in the absence or presence of 20 ng pcDNA3-MK2-WT, 20 ng pcDNA3-MK2-K76R) or 20 μM SB203580. 6 replicates of each combination were measured. Graphs represent mean (± SD) luciferase activity in corrected for Renilla luciferase activity.

    Journal: Molecular cancer research : MCR

    Article Title: The p38 MAPK-MK2 axis regulates E2F1 and FOXM1 expression after epirubicin treatment

    doi: 10.1158/1541-7786.MCR-11-0559

    Figure Lengend Snippet: FOXM1 phosphorylation is responsible for the induction of E2F1 protein expression in response to epirubicin A. Neither Ser-364 nor Ser-31 is solely responsible for the induction of E2F1 protein expression in response to epirubicin . U2OS cells were transfected with either the empty pCMV plasmid, pCMV-E2F1-132E, wild-type pCMV-HA-E2F1 plasmid, or an pCMV-HA-E2F1 mutant plasmid bearing one of the following point mutations: S31A, S31D, S364A or S364D. Thirty–six h later transfected cells were either untreated or exposed to epirubicin for 6 h and harvested. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the antibodies indicated. B. E2F1 phosphorylation mutants have depleted transactivation activity . Four thousand cells were seeded in each well of a 96-well plate, allowed to attach overnight and transfected with 20 ng of the indicated expression plasmids together with 5ng Renilla reporter and 20 ng of the luciferase reporter gene driven by the E2F1 promoter. Twenty h later, 6 replicates of each condition were left untreated while 6 others were treated with 1 μM epirubicin for 3 h prior to measurement of luciferase activity. Graphs represent mean (± SD) luciferase activity corrected for Renilla luciferase activity. Values were normalised to that of the empty vector in untreated cells. C. S364-phosphorylation contributes to E2F1 activation . 4000 cells were seeded in 96-well plates, allowed to attach overnight and transfected with either empty vector, pCMV-E2F1-132E or increasing concentrations (0, 10, 20 or 30 ng) of wild-type HA-E2F1 or the E2F1-S364A point mutant together with 5 ng Renilla reporter and 20 ng of the luciferase reporter gene driven by the E2F1 promoter. 6 replicates of each combination were measured. Graphs represent mean (± SD) luciferase activity in corrected for Renilla luciferase activity. Values were normalised to that of empty vector (-). D. MK2 and p38 contribute to FOXM1 transactivation by E2F1 . U2OS cells were transfected with 20 ng of either empty vector, pCMV-E2F1-132E or wild-type HA-E2F1 together with 5 ng Renilla reporter and 20 ng of the luciferase reporter gene driven by the FOXM1 ) in the absence or presence of 20 ng pcDNA3-MK2-WT, 20 ng pcDNA3-MK2-K76R) or 20 μM SB203580. 6 replicates of each combination were measured. Graphs represent mean (± SD) luciferase activity in corrected for Renilla luciferase activity.

    Article Snippet: The inhibitors SB203580 and SP600125 were acquired from Calbiochem (Merck), while SB202190 was from Sigma.

    Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Activity Assay, Luciferase, Activation Assay

    JNK inhibition attenuates while p38 inhibitor enhances epirubicin-induced apoptosis A. JNK inhibition attenuates epirubicin-induced apoptosis, whereas p38 inhibition sensitizes U2OS cells to epirubicin-induced apoptosis . U2OS cells were either untreated or pretreated with either 25 μM SP600125 or 15 μM SB202190 during 1h prior to adding epirubicin to the media (final concentration 1μM). Samples were collected by trypsinization 0, 6 or 24 h after epirubicin addition, stained with propidium iodide, and analyzed by flow cytometry to determine the cell cycle distribution (upper panels). The chart below shows the quantitation of the percentage of cells with sub-G1 DNA content. B. Opposing effects of p38 and JNK inhibition on epirubicin-induced FOXM1 upregulation and apoptotic cell death . U2OS cells were either pretreated with 20 μM SB203580 or 25 μM SP600125 or untreated and 1h later exposed to 1 μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted from lysates. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. C. Absence of JNK in MEFs augments the induction of FOXM1 expression by epirubicin and suppresses epirubicin-induced apoptosis . Wild-type, JNK1 -/- JNK2 -/- MEFs were treated with 1μ epirubicin for 0, 4 or 8 h. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control. D. JNK depletion augments E2F1 and FOXM1 levels induced by epirubicin treatment and confers resistance to epirubicin-induced apoptosis . U2OS cells were transfected with nontargeting siRNA or siRNAs specific to either JNK1 and JNK2. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control.

    Journal: Molecular cancer research : MCR

    Article Title: The p38 MAPK-MK2 axis regulates E2F1 and FOXM1 expression after epirubicin treatment

    doi: 10.1158/1541-7786.MCR-11-0559

    Figure Lengend Snippet: JNK inhibition attenuates while p38 inhibitor enhances epirubicin-induced apoptosis A. JNK inhibition attenuates epirubicin-induced apoptosis, whereas p38 inhibition sensitizes U2OS cells to epirubicin-induced apoptosis . U2OS cells were either untreated or pretreated with either 25 μM SP600125 or 15 μM SB202190 during 1h prior to adding epirubicin to the media (final concentration 1μM). Samples were collected by trypsinization 0, 6 or 24 h after epirubicin addition, stained with propidium iodide, and analyzed by flow cytometry to determine the cell cycle distribution (upper panels). The chart below shows the quantitation of the percentage of cells with sub-G1 DNA content. B. Opposing effects of p38 and JNK inhibition on epirubicin-induced FOXM1 upregulation and apoptotic cell death . U2OS cells were either pretreated with 20 μM SB203580 or 25 μM SP600125 or untreated and 1h later exposed to 1 μM epirubicin. At the times indicated at the top of each lane cells were harvested and proteins extracted from lysates. Equal amounts of protein were used for Western blot analysis with the indicated antibodies. Anti-β-tubulin was immunoblotted as a loading control. C. Absence of JNK in MEFs augments the induction of FOXM1 expression by epirubicin and suppresses epirubicin-induced apoptosis . Wild-type, JNK1 -/- JNK2 -/- MEFs were treated with 1μ epirubicin for 0, 4 or 8 h. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control. D. JNK depletion augments E2F1 and FOXM1 levels induced by epirubicin treatment and confers resistance to epirubicin-induced apoptosis . U2OS cells were transfected with nontargeting siRNA or siRNAs specific to either JNK1 and JNK2. 48h later, cells were treated with 1μM epirubicin and harvested at the indicated time points. Whole cell extracts were prepared and equal amounts of protein were subjected to Western blot analysis using the indicated antiobodies. Anti-β-tubulin was immunoblotted as a loading control.

    Article Snippet: The inhibitors SB203580 and SP600125 were acquired from Calbiochem (Merck), while SB202190 was from Sigma.

    Techniques: Inhibition, Concentration Assay, Staining, Flow Cytometry, Cytometry, Quantitation Assay, Western Blot, Expressing, Transfection

    AL-LC induction of STC1 is p38MAPK dependent in vitro and in vivo. a AL-LC induced upregulation of STC1 protein expression in cardiomyocytes was abolished in the presence of p38MAPK inhibitor, SB203580 (5 μmol/L). b AL-LC triggered upregulation

    Journal: Basic research in cardiology

    Article Title: Stanniocalcin1 is a key mediator of amyloidogenic light chain induced cardiotoxicity

    doi: 10.1007/s00395-013-0378-5

    Figure Lengend Snippet: AL-LC induction of STC1 is p38MAPK dependent in vitro and in vivo. a AL-LC induced upregulation of STC1 protein expression in cardiomyocytes was abolished in the presence of p38MAPK inhibitor, SB203580 (5 μmol/L). b AL-LC triggered upregulation

    Article Snippet: SB203580 compound was obtained from Calbiochem.

    Techniques: In Vitro, In Vivo, Expressing

    SAA -1 induces IL-10 production from human neutrophils ( a ). IL-10 secretion by SAA-1 treated CD11b + CD15 + cells purified from healthy donors in response to increasing concentrations of SAA-1 . ( b ) IL-10 secretion by CD11b + CD15 + cells from healthy donors, treated with SAA-1, Pam3Cys or FMLP in the presence or absence of blocking FPR-2 and TLR-2 antibodies. ( c ) T cell proliferation of alloreactive T cells stimulated by allogeneic DC, with graded numbers of CD11b + CD15 + cells from third party healthy donors pretreated or not with SAA-1 (100 % proliferation corresponds to 7 × 10 4 cpm). ( d ) IL-10 release from CD11b + CD15 + cells purified from the melanoma patient △ (as shown in Supplementary Fig. 2a ) (top panel), and one healthy donor (bottom panel) pre-incubated or not with the PI(3) kinase inhibitor LY294002, the Erk inhibitor U1026 and the p38 inhibitor SB203580 and then treated or not with SAA-1. Data are representative of five independent experiments (error bars, SD).

    Journal: Nature immunology

    Article Title: Invariant NKT cells modulate the suppressive activity of Serum Amyloid A-differentiated IL-10-secreting neutrophils

    doi: 10.1038/ni.1942

    Figure Lengend Snippet: SAA -1 induces IL-10 production from human neutrophils ( a ). IL-10 secretion by SAA-1 treated CD11b + CD15 + cells purified from healthy donors in response to increasing concentrations of SAA-1 . ( b ) IL-10 secretion by CD11b + CD15 + cells from healthy donors, treated with SAA-1, Pam3Cys or FMLP in the presence or absence of blocking FPR-2 and TLR-2 antibodies. ( c ) T cell proliferation of alloreactive T cells stimulated by allogeneic DC, with graded numbers of CD11b + CD15 + cells from third party healthy donors pretreated or not with SAA-1 (100 % proliferation corresponds to 7 × 10 4 cpm). ( d ) IL-10 release from CD11b + CD15 + cells purified from the melanoma patient △ (as shown in Supplementary Fig. 2a ) (top panel), and one healthy donor (bottom panel) pre-incubated or not with the PI(3) kinase inhibitor LY294002, the Erk inhibitor U1026 and the p38 inhibitor SB203580 and then treated or not with SAA-1. Data are representative of five independent experiments (error bars, SD).

    Article Snippet: Kinase inhibitors were SB203580 (p38), U1026 (Erk) and LY294002 (PI(3)K) (from Calbiochem Merck).

    Techniques: Purification, Blocking Assay, Incubation

    The inhibition of p38MAPK and NF-κB activities stimulated by aldosterone (Aldo) in rat VSMCs. The cells were pretreated with SB203580 (SB, 20 μmol/L), TLCK (100 μmol/L), and spironolactone (Spir, 50 μmol/L) for 1 h, then stimulated with Aldo (0.1 μmol/L) for 4 h. The cell lysates were analysed by Western blotting with specific antibodies. Mean±SD. n =3. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells

    doi: 10.1038/aps.2012.36

    Figure Lengend Snippet: The inhibition of p38MAPK and NF-κB activities stimulated by aldosterone (Aldo) in rat VSMCs. The cells were pretreated with SB203580 (SB, 20 μmol/L), TLCK (100 μmol/L), and spironolactone (Spir, 50 μmol/L) for 1 h, then stimulated with Aldo (0.1 μmol/L) for 4 h. The cell lysates were analysed by Western blotting with specific antibodies. Mean±SD. n =3. b P

    Article Snippet: Materials Aldosterone, spironolactone, α-p-tosyl-L -lysine chloromethyl ketone (TLCK), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA); SB203580 (SB) and PD98059 (PD) were from Calbiochem (Darmstadt, Germany).

    Techniques: Inhibition, Western Blot

    The expression of IL-6 in VSMCs induced by aldosterone (Aldo). The cells were pretreated with SB203580 (SB, 20 μmol/L), spironolactone (Spir, 50 μmol/L) and TLCK (100 μmol/L) for 1 h, then stimulated with Aldo (0.1 μmol/L) for 7 h. The cell lysates were analysed by real time RT-PCR. Mean±SD. n =3. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells

    doi: 10.1038/aps.2012.36

    Figure Lengend Snippet: The expression of IL-6 in VSMCs induced by aldosterone (Aldo). The cells were pretreated with SB203580 (SB, 20 μmol/L), spironolactone (Spir, 50 μmol/L) and TLCK (100 μmol/L) for 1 h, then stimulated with Aldo (0.1 μmol/L) for 7 h. The cell lysates were analysed by real time RT-PCR. Mean±SD. n =3. b P

    Article Snippet: Materials Aldosterone, spironolactone, α-p-tosyl-L -lysine chloromethyl ketone (TLCK), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA); SB203580 (SB) and PD98059 (PD) were from Calbiochem (Darmstadt, Germany).

    Techniques: Expressing, Quantitative RT-PCR

    Aldosterone (Aldo) stimulates the phosphorylation of p38MAPK (A) and ERK1/2 (B) in VSMCs. The cells were pretreated with SB203580 (SB, 20 μmol/L), PD98059 (PD, 50 μmol/L), and spironolactone (Spir, 50 μmol/L) for 1 h, then stimulated with Aldo (0.1 μmol/L) for 30 min. The cell lysates were analysed by Western blotting with specific antibodies.

    Journal: Acta Pharmacologica Sinica

    Article Title: The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells

    doi: 10.1038/aps.2012.36

    Figure Lengend Snippet: Aldosterone (Aldo) stimulates the phosphorylation of p38MAPK (A) and ERK1/2 (B) in VSMCs. The cells were pretreated with SB203580 (SB, 20 μmol/L), PD98059 (PD, 50 μmol/L), and spironolactone (Spir, 50 μmol/L) for 1 h, then stimulated with Aldo (0.1 μmol/L) for 30 min. The cell lysates were analysed by Western blotting with specific antibodies.

    Article Snippet: Materials Aldosterone, spironolactone, α-p-tosyl-L -lysine chloromethyl ketone (TLCK), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA); SB203580 (SB) and PD98059 (PD) were from Calbiochem (Darmstadt, Germany).

    Techniques: Western Blot

    The expression of cyclooxyenase 2 (Cox-2) in rat vascular smooth muscle cells (VSMCs) induced by aldosterone (Aldo). (A) Upper panel, the concentration-dependent increase in Cox-2 expression induced by Aldo for 4 h. Lower panel, the time-related increase in Cox-2 expression induced by Aldo (0.1 μmol/L) for the indicated times. (B) The effects of different inhibitors on Cox-2 expression induced by Aldo. The cells were pretreated with SB203580 (SB, 20 μmol/L), PD98059 (PD, 50 μmol/L), and spironolactone (Spir, 50 μmol/L) for 1 h, then stimulated by Aldo (0.1 μmol/L) for 4 h. The cell lysates were analysed by Western blotting with specific antibodies. Mean±SD. n =3. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells

    doi: 10.1038/aps.2012.36

    Figure Lengend Snippet: The expression of cyclooxyenase 2 (Cox-2) in rat vascular smooth muscle cells (VSMCs) induced by aldosterone (Aldo). (A) Upper panel, the concentration-dependent increase in Cox-2 expression induced by Aldo for 4 h. Lower panel, the time-related increase in Cox-2 expression induced by Aldo (0.1 μmol/L) for the indicated times. (B) The effects of different inhibitors on Cox-2 expression induced by Aldo. The cells were pretreated with SB203580 (SB, 20 μmol/L), PD98059 (PD, 50 μmol/L), and spironolactone (Spir, 50 μmol/L) for 1 h, then stimulated by Aldo (0.1 μmol/L) for 4 h. The cell lysates were analysed by Western blotting with specific antibodies. Mean±SD. n =3. b P

    Article Snippet: Materials Aldosterone, spironolactone, α-p-tosyl-L -lysine chloromethyl ketone (TLCK), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA); SB203580 (SB) and PD98059 (PD) were from Calbiochem (Darmstadt, Germany).

    Techniques: Expressing, Concentration Assay, Western Blot

    p38MAPK and Ca ++ /CaMK pathways mediate CREB phosphorylation induced by Ang II. Representative immunoblots showing the effect of (A) pretreatment with SB203580 (10 µM) followed by Ang II (100 nM) activation on p-CREB levels over a specified time course. (B) pretreatment with KN62 (10 µM) followed by Ang II (100 nM) at 1 hour on the phosphorylation of CREB. (C) CATH.a cells were preincubated for 30 minutes with PD98059 (25 µM), SB203580 (20 µM) and KN62 (10 µM) followed by Ang II (100 nM) for 8 hours. Cell lysates were immunoblotted for p-CREB. (D) Cells were preincubated with or without Losartan (1 µM) followed by Ang II (100 nM) for 8 hours. At the end of the specified time period, cell lysates were tested for p-CREB levels by western blot. *p

    Journal: PLoS ONE

    Article Title: NF-?B and CREB Are Required for Angiotensin II Type 1 Receptor Upregulation in Neurons

    doi: 10.1371/journal.pone.0078695

    Figure Lengend Snippet: p38MAPK and Ca ++ /CaMK pathways mediate CREB phosphorylation induced by Ang II. Representative immunoblots showing the effect of (A) pretreatment with SB203580 (10 µM) followed by Ang II (100 nM) activation on p-CREB levels over a specified time course. (B) pretreatment with KN62 (10 µM) followed by Ang II (100 nM) at 1 hour on the phosphorylation of CREB. (C) CATH.a cells were preincubated for 30 minutes with PD98059 (25 µM), SB203580 (20 µM) and KN62 (10 µM) followed by Ang II (100 nM) for 8 hours. Cell lysates were immunoblotted for p-CREB. (D) Cells were preincubated with or without Losartan (1 µM) followed by Ang II (100 nM) for 8 hours. At the end of the specified time period, cell lysates were tested for p-CREB levels by western blot. *p

    Article Snippet: The following inhibitors were used to determine the specific upstream molecular pathways: SB203580 (p38MAPK), KN-62 (Ca2+ /calmodulin-dependent protein kinase II), losartan (AT1 R) (Sigma-Aldrich), PD98059 (ERK) (Cell Signaling Technology, Danvers, MA).

    Techniques: Western Blot, Activation Assay

    UV-induced cell death is JNK dependent. A , Dusp1 / Mkp-1 −/− MEFs were pre-treated with dimethyl sulfoxide ( DMSO ) or 10 μ m SB203580, and then exposed to 30 J/m 2 UV. Following incubation at 37 °C for 1 h, cells were lysed, and proteins analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. B , Dusp1 / Mkp-1 −/− MEFs were treated as in A . After 36 h, cell survival was assessed by MTS assay and the results normalized to untreated control values. C , Dusp1 / Mkp-1 −/− MEFs were transfected with either empty vector or an expression vector encoding either wild-type ( WT ) MKP-1 or MKP-1 M. After 24 h, cells were exposed to 30 J/m 2 UV and incubated at 37 °C for 1 h. Cells were then lysed, and proteins analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. D , Dusp1 / Mkp-1 −/− MEFs were transfected as in C . After 24 h, cells were exposed to 30 J/m 2 UV. Following incubation at 37 °C for 36 h, cell survival was assessed by MTS assay, and the results were normalized to untreated control values. * = p

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between the p38? and JNK MAPK Pathways Mediated by MAP Kinase Phosphatase-1 Determines Cellular Sensitivity to UV Radiation *

    doi: 10.1074/jbc.M110.117911

    Figure Lengend Snippet: UV-induced cell death is JNK dependent. A , Dusp1 / Mkp-1 −/− MEFs were pre-treated with dimethyl sulfoxide ( DMSO ) or 10 μ m SB203580, and then exposed to 30 J/m 2 UV. Following incubation at 37 °C for 1 h, cells were lysed, and proteins analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. B , Dusp1 / Mkp-1 −/− MEFs were treated as in A . After 36 h, cell survival was assessed by MTS assay and the results normalized to untreated control values. C , Dusp1 / Mkp-1 −/− MEFs were transfected with either empty vector or an expression vector encoding either wild-type ( WT ) MKP-1 or MKP-1 M. After 24 h, cells were exposed to 30 J/m 2 UV and incubated at 37 °C for 1 h. Cells were then lysed, and proteins analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. D , Dusp1 / Mkp-1 −/− MEFs were transfected as in C . After 24 h, cells were exposed to 30 J/m 2 UV. Following incubation at 37 °C for 36 h, cell survival was assessed by MTS assay, and the results were normalized to untreated control values. * = p

    Article Snippet: Reagents The inhibitor SB203580 was purchased from Calbiochem.

    Techniques: Incubation, SDS Page, Western Blot, MTS Assay, Transfection, Plasmid Preparation, Expressing

    Induction of DUSP1/MKP-1 by UV is dependent on signaling via MSK1/2 and CREB/ATF1. A , wild-type MEFs were transfected with either a control siRNA, or siRNAs targeting MSK1 and MSK2. Cells were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. Cells were then lysed and either DUSP1/MKP-1 mRNA levels were determined using quantitative RT-PCR ( left ) or proteins were analyzed by SDS-PAGE and Western blotting using the indicated antibodies ( right ). B , wild-type MEFs were transfected with either empty vector or an expression vector ( Vec ) encoding hemagglutinin-tagged A-CREB. After 24 h, cells were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. Cells were then lysed and either DUSP1/MKP-1 mRNA levels were determined using quantitative RT-PCR ( left ) or proteins were analyzed by SDS-PAGE and Western blotting using the indicated antibodies ( right ). Wild-type MEFs stably transfected with the MKP-1 reporter pGL4.28-MKP-1-luc were either pre-treated for 1 h with dimethyl sulfoxide ( DMSO ) or 10 μ m SB203580 ( C ), transfected with either empty vector or an expression vector encoding A-CREB ( D ), or transfected with either a control siRNA or siRNAs targeting MSK1 and -2 ( E ). Cells were then either mock irradiated or exposed to 30 J/m 2 UV and incubated at 37 °C for 8 h. Cells were then lysed, firefly luciferase activity was quantified and its levels normalized to protein concentration. Experiments were performed three times, each with triplicate determinations, and mean values with associated errors are shown (mean ± S.E.). DMSO , dimethyl sulfoxide; HA , hemagglutinin.

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between the p38? and JNK MAPK Pathways Mediated by MAP Kinase Phosphatase-1 Determines Cellular Sensitivity to UV Radiation *

    doi: 10.1074/jbc.M110.117911

    Figure Lengend Snippet: Induction of DUSP1/MKP-1 by UV is dependent on signaling via MSK1/2 and CREB/ATF1. A , wild-type MEFs were transfected with either a control siRNA, or siRNAs targeting MSK1 and MSK2. Cells were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. Cells were then lysed and either DUSP1/MKP-1 mRNA levels were determined using quantitative RT-PCR ( left ) or proteins were analyzed by SDS-PAGE and Western blotting using the indicated antibodies ( right ). B , wild-type MEFs were transfected with either empty vector or an expression vector ( Vec ) encoding hemagglutinin-tagged A-CREB. After 24 h, cells were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. Cells were then lysed and either DUSP1/MKP-1 mRNA levels were determined using quantitative RT-PCR ( left ) or proteins were analyzed by SDS-PAGE and Western blotting using the indicated antibodies ( right ). Wild-type MEFs stably transfected with the MKP-1 reporter pGL4.28-MKP-1-luc were either pre-treated for 1 h with dimethyl sulfoxide ( DMSO ) or 10 μ m SB203580 ( C ), transfected with either empty vector or an expression vector encoding A-CREB ( D ), or transfected with either a control siRNA or siRNAs targeting MSK1 and -2 ( E ). Cells were then either mock irradiated or exposed to 30 J/m 2 UV and incubated at 37 °C for 8 h. Cells were then lysed, firefly luciferase activity was quantified and its levels normalized to protein concentration. Experiments were performed three times, each with triplicate determinations, and mean values with associated errors are shown (mean ± S.E.). DMSO , dimethyl sulfoxide; HA , hemagglutinin.

    Article Snippet: Reagents The inhibitor SB203580 was purchased from Calbiochem.

    Techniques: Transfection, Irradiation, Incubation, Quantitative RT-PCR, SDS Page, Western Blot, Plasmid Preparation, Expressing, Stable Transfection, Luciferase, Activity Assay, Protein Concentration

    Loss of DUSP1/MKP-1 results in increased MAPK signaling and gene transcription. A , wild-type ( WT ) or Dusp1 / Mkp-1 −/− MEFs were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. c-Fos mRNA levels were determined using quantitative RT-PCR. B , wild-type or Dusp1 / Mkp-1 −/− MEFs were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. Cells were lysed, and proteins analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. C , wild-type or Dusp1 / Mkp-1 −/− MEFs were cotransfected with a firefly luciferase reporter driven by the proximal 3-kb region of the DUSP1 / MKP-1 promoter and as a transfection control a plasmid encoding Renilla luciferase. After 24 h, cells were preincubated with either dimethyl sulfoxide ( DMSO ) or 10 μ m SB203580 ( SB ), exposed to 30 J/m 2 UV, and then incubated at 37 °C for 5 h. Cells were lysed, and luciferase activities were determined. Experiments were performed three times, each with triplicate determinations and mean values with associated errors are shown (mean ± S.E.).

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between the p38? and JNK MAPK Pathways Mediated by MAP Kinase Phosphatase-1 Determines Cellular Sensitivity to UV Radiation *

    doi: 10.1074/jbc.M110.117911

    Figure Lengend Snippet: Loss of DUSP1/MKP-1 results in increased MAPK signaling and gene transcription. A , wild-type ( WT ) or Dusp1 / Mkp-1 −/− MEFs were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. c-Fos mRNA levels were determined using quantitative RT-PCR. B , wild-type or Dusp1 / Mkp-1 −/− MEFs were either mock irradiated or exposed to 30 J/m 2 UV and then incubated at 37 °C for the indicated times. Cells were lysed, and proteins analyzed by SDS-PAGE followed by Western blotting using the indicated antibodies. C , wild-type or Dusp1 / Mkp-1 −/− MEFs were cotransfected with a firefly luciferase reporter driven by the proximal 3-kb region of the DUSP1 / MKP-1 promoter and as a transfection control a plasmid encoding Renilla luciferase. After 24 h, cells were preincubated with either dimethyl sulfoxide ( DMSO ) or 10 μ m SB203580 ( SB ), exposed to 30 J/m 2 UV, and then incubated at 37 °C for 5 h. Cells were lysed, and luciferase activities were determined. Experiments were performed three times, each with triplicate determinations and mean values with associated errors are shown (mean ± S.E.).

    Article Snippet: Reagents The inhibitor SB203580 was purchased from Calbiochem.

    Techniques: Irradiation, Incubation, Quantitative RT-PCR, SDS Page, Western Blot, Luciferase, Transfection, Plasmid Preparation

    Effects of p38 inhibition on the protective role of resveratrol on PMVECs. PMVECs were exposed to 10% sham serum (SS), 10% SS + resveratrol (resv) (20 μM), 10% SS + SB203580 (20 μM), 10% burn serum (BS), 10% BS + resv, 10% BS + SB203580, 10% BS + resv + SB203580 for 12 h. ( A ) Flow cytometry analysis showing the percentage of apoptotic cells in each treatment group. ( B ) Representative immunoblots showing the protein level changes of p -p38 in each treatment group. ( C ) Representative immunoblots showing the protein level change of cleaved caspase-3 in each treatment group. Results represent mean ± SEM of six independent experiments using different batches of PMVECs from different rats. * p

    Journal: Scientific Reports

    Article Title: SIRT1 protects rat lung tissue against severe burn-induced remote ALI by attenuating the apoptosis of PMVECs via p38 MAPK signaling

    doi: 10.1038/srep10277

    Figure Lengend Snippet: Effects of p38 inhibition on the protective role of resveratrol on PMVECs. PMVECs were exposed to 10% sham serum (SS), 10% SS + resveratrol (resv) (20 μM), 10% SS + SB203580 (20 μM), 10% burn serum (BS), 10% BS + resv, 10% BS + SB203580, 10% BS + resv + SB203580 for 12 h. ( A ) Flow cytometry analysis showing the percentage of apoptotic cells in each treatment group. ( B ) Representative immunoblots showing the protein level changes of p -p38 in each treatment group. ( C ) Representative immunoblots showing the protein level change of cleaved caspase-3 in each treatment group. Results represent mean ± SEM of six independent experiments using different batches of PMVECs from different rats. * p

    Article Snippet: Drug Administration in PMVECs For detecting SIRT1 expression and the activities of caspase-3 and MAPKs, approximately 2.0 × 106 serum-starved PMVECs were seeded into 21-cm2 dishes, treated with 10% (vol/vol in culture medium) sham serum (SS), 10% SS + resv (20 μM), 10% SS + SB203580 (20 μM) (Sigma, St. Louis, MO), 10% burn serum (BS), 10% BS + resv, 10% BS + resv + EX527 (1 μM) (Sigma, St. Louis, MO), or 10% BS + resv + SB203580 for 12 h. For SIRT1 silencing, PMVECs at the same density as above were transfected with either SIRT1 shRNA or NT shRNA for 48 h and then stimulated by 10% SS or 10% BS for 12 h. Cells were then collected for Western blotting, quantitative real-time PCR and flow cytometry analysis.

    Techniques: Inhibition, Flow Cytometry, Cytometry, Western Blot

    JNK specifically phosphorylates YAP in vivo . ( a ) HaCaT, U2OS, MCF7 and 293T cell lines were treated with anisomycin or DMSO (control) for 60 min before harvesting. YAP band shift was analyzed as described before and lysates were analyzed by Western blotting with the indicated antibodies. ( b ) Upper panel: U2OS cells were treated with SP600125 or an equivalent volume of DMSO for 3 h, followed by treatment for 15 min with anisomycin or DMSO. Lower panel: HaCaT cells were treated with SB203580 or DMSO for 3 h prior followed by a 15 min treatment with anisomycin or DMSO, harvested and lysates were analyzed using the indicated antibodies. ( c ) U2OS cells were transfected with Flag–YAP and either siControl (siCTL) or siJNK1 and siJNK2 (siJNK). The cells were then treated with anisomycin for 1 h and subsequently Flag–YAP was immunoprecipitated from lysates. IP inputs and eluates were analyzed by Western blotting using the indicated antibodies. ( d ) BWT cells were transfected with Flag–YAP, Flag–mut2AYAP and Flag–mut2DYAP. The cells were irradiated with 30 J/m 2 UV-C and harvested 1 h later for Western blot analysis with the indicated antibodies. ( e ) HaCaT cells were irradiated with 50 J/m 2 UV-C and harvested at 10, 20, 30 min and 2 h. Endogenous YAP was immunoprecipitated and IP inputs and eluates were analyzed by Western blotting using the indicated antibodies

    Journal: Cell Death & Disease

    Article Title: JNK phosphorylates Yes-associated protein (YAP) to regulate apoptosis

    doi: 10.1038/cddis.2010.7

    Figure Lengend Snippet: JNK specifically phosphorylates YAP in vivo . ( a ) HaCaT, U2OS, MCF7 and 293T cell lines were treated with anisomycin or DMSO (control) for 60 min before harvesting. YAP band shift was analyzed as described before and lysates were analyzed by Western blotting with the indicated antibodies. ( b ) Upper panel: U2OS cells were treated with SP600125 or an equivalent volume of DMSO for 3 h, followed by treatment for 15 min with anisomycin or DMSO. Lower panel: HaCaT cells were treated with SB203580 or DMSO for 3 h prior followed by a 15 min treatment with anisomycin or DMSO, harvested and lysates were analyzed using the indicated antibodies. ( c ) U2OS cells were transfected with Flag–YAP and either siControl (siCTL) or siJNK1 and siJNK2 (siJNK). The cells were then treated with anisomycin for 1 h and subsequently Flag–YAP was immunoprecipitated from lysates. IP inputs and eluates were analyzed by Western blotting using the indicated antibodies. ( d ) BWT cells were transfected with Flag–YAP, Flag–mut2AYAP and Flag–mut2DYAP. The cells were irradiated with 30 J/m 2 UV-C and harvested 1 h later for Western blot analysis with the indicated antibodies. ( e ) HaCaT cells were irradiated with 50 J/m 2 UV-C and harvested at 10, 20, 30 min and 2 h. Endogenous YAP was immunoprecipitated and IP inputs and eluates were analyzed by Western blotting using the indicated antibodies

    Article Snippet: The SP600125 and SB203580 inhibitors (Calbiochem, Merck Chemicals Ltd., Nottingham, UK) were used for 3 h before 15-min anisomycin treatment at a concentration of 20 and 10 μ M, respectively.

    Techniques: In Vivo, Electrophoretic Mobility Shift Assay, Western Blot, Transfection, Immunoprecipitation, Irradiation

    MAPK-mediated phosphorylation of Mnk1a at T209/T214 activates Mnk1-eIF4G interaction. (A) HEK-293 eIF4G cells were uninduced or Tet induced, serum starved for 18 h, and treated for 15 min with DMSO, 20% fetal bovine serum (FBS), 100 nM TPA (Erk1/2 stimulation), 0.1 mM sodium arsenite (ars; broad stimulation), or 10 μg/ml anisomycin (aniso; p38 stimulation). Cell extracts were subjected to anti-Flag IP. (B) Schematic representation of MAPK signal transduction to eIF4E and corresponding kinase inhibitors. (C and D) HEK-293 eIF4G cells were serum starved for 18 h, incubated for 2 h with DMSO, 20 μM UO126 (MEK inhibitor), or 10 μM SB203580 (p38 inhibitor), and treated for 15 min with DMSO (−), 100 nM TPA, or 10 μg/ml anisomycin. Cell extracts were subjected to anti-Flag IP. (E) HEK-293 eIF4G cells were transfected with the indicated HA-Mnk1a variants for 24 h and stimulated for 15 min with DMSO (−) or 100 nM TPA (+). Cell extracts were subjected to anti-Flag IP. (F) HEK-293 cells were transfected with the indicated HA-Mnk1a variants for 24 h and treated for 15 min with 100 nM TPA (+). Cell extracts were subjected to anti-HA IP.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Eukaryotic Initiation Factor 4E (eIF4E) Phosphorylation by Mitogen-Activated Protein Kinase Occurs through Modulation of Mnk1-eIF4G Interaction ▿

    doi: 10.1128/MCB.00448-10

    Figure Lengend Snippet: MAPK-mediated phosphorylation of Mnk1a at T209/T214 activates Mnk1-eIF4G interaction. (A) HEK-293 eIF4G cells were uninduced or Tet induced, serum starved for 18 h, and treated for 15 min with DMSO, 20% fetal bovine serum (FBS), 100 nM TPA (Erk1/2 stimulation), 0.1 mM sodium arsenite (ars; broad stimulation), or 10 μg/ml anisomycin (aniso; p38 stimulation). Cell extracts were subjected to anti-Flag IP. (B) Schematic representation of MAPK signal transduction to eIF4E and corresponding kinase inhibitors. (C and D) HEK-293 eIF4G cells were serum starved for 18 h, incubated for 2 h with DMSO, 20 μM UO126 (MEK inhibitor), or 10 μM SB203580 (p38 inhibitor), and treated for 15 min with DMSO (−), 100 nM TPA, or 10 μg/ml anisomycin. Cell extracts were subjected to anti-Flag IP. (E) HEK-293 eIF4G cells were transfected with the indicated HA-Mnk1a variants for 24 h and stimulated for 15 min with DMSO (−) or 100 nM TPA (+). Cell extracts were subjected to anti-Flag IP. (F) HEK-293 cells were transfected with the indicated HA-Mnk1a variants for 24 h and treated for 15 min with 100 nM TPA (+). Cell extracts were subjected to anti-HA IP.

    Article Snippet: Inhibitors of MEK (UO126) (Cell Signaling, Danvers, MA), Mnk ( ) (Sigma, St. Louis, MO), and p38 (SB203580) (Sigma) were dissolved in dimethyl sulfoxide (DMSO) and used at the concentrations indicated.

    Techniques: Transduction, Incubation, Transfection

    JNK and p38 stress MAPK pathways dowregulate Mss4 expression. A7 melanoma cells were co-transfected with reporter construct driven by the 3.6-kb Mss4 promoter fragment and different c.a. or d.n. members of the p38 ( a ) or JNK ( b ) signaling pathways. The SB203580 ( c ) and SP600125 inhibitors ( d ) were added 24 h after transfection with c.a. MKK6 ( c ) and MKK7 ( d ) constructs. Reporter gene activity was determined 48 h later. Values of control cells transfected with reporter gene construct plus empty vector were taken as unity. Mean values±S.D. from three repeated experiments are shown. * P

    Journal: Cell Death & Disease

    Article Title: Mss4 protein is a regulator of stress response and apoptosis

    doi: 10.1038/cddis.2012.37

    Figure Lengend Snippet: JNK and p38 stress MAPK pathways dowregulate Mss4 expression. A7 melanoma cells were co-transfected with reporter construct driven by the 3.6-kb Mss4 promoter fragment and different c.a. or d.n. members of the p38 ( a ) or JNK ( b ) signaling pathways. The SB203580 ( c ) and SP600125 inhibitors ( d ) were added 24 h after transfection with c.a. MKK6 ( c ) and MKK7 ( d ) constructs. Reporter gene activity was determined 48 h later. Values of control cells transfected with reporter gene construct plus empty vector were taken as unity. Mean values±S.D. from three repeated experiments are shown. * P

    Article Snippet: The inhibition of p38- and JNK-stress cascades was accomplished by treatment with 20 μ M SB203580 and 10 μ M SP600125, respectively (Calbiochem, Nottingham, UK).

    Techniques: Expressing, Transfection, Construct, Activity Assay, Plasmid Preparation

    p38 is involved in the quercetin-induced ABCA1 expression and cholesterol efflux. RAW264.7 macrophages were pretreated with 20 μM SB203580 (p38 inhibitor), and then treated with 100 μM quercetin. A: The ABCA1 mRNA levels were measured by quantitative real-time PCR and normalized to GAPDH. B: The ABCA1 protein levels were detected by Western blot analysis and α-tubulin was utilized as a loading control. The normalized level of mRNA or protein from cells without quercetin treatment was set as 1. RAW264.7 macrophages were infected with lentivirus expressing p38 shRNA-1 or p38 shRNA-2 to confirm the effects of p38 on quercetin-induced ABCA1 expression and cholesterol efflux. Luciferase shRNA (Luc. shRNA) was used as a control shRNA. C: The knockdown efficiency of p38 was checked by Western blot analysis. D, E: The effect of p38 knockdown on quercetin-induced ABCA1 mRNA (D) and protein (E) expression was examined by quantitative real-time PCR and Western blot analysis, respectively. The normalized level of mRNA or protein from parental cells without quercetin treatment was given as 1. F: Cholesterol efflux from p38 knockdown cells to media was measured in the presence of 10 μg/ml apoAI. Cholesterol efflux was expressed as the percentage of radioactivity in the medium relative to the total radioactivity (medium and cells). Values are mean ± SEM (n = 3–6). * P

    Journal: Journal of Lipid Research

    Article Title: Quercetin enhances ABCA1 expression and cholesterol efflux through a p38-dependent pathway in macrophages

    doi: 10.1194/jlr.M024471

    Figure Lengend Snippet: p38 is involved in the quercetin-induced ABCA1 expression and cholesterol efflux. RAW264.7 macrophages were pretreated with 20 μM SB203580 (p38 inhibitor), and then treated with 100 μM quercetin. A: The ABCA1 mRNA levels were measured by quantitative real-time PCR and normalized to GAPDH. B: The ABCA1 protein levels were detected by Western blot analysis and α-tubulin was utilized as a loading control. The normalized level of mRNA or protein from cells without quercetin treatment was set as 1. RAW264.7 macrophages were infected with lentivirus expressing p38 shRNA-1 or p38 shRNA-2 to confirm the effects of p38 on quercetin-induced ABCA1 expression and cholesterol efflux. Luciferase shRNA (Luc. shRNA) was used as a control shRNA. C: The knockdown efficiency of p38 was checked by Western blot analysis. D, E: The effect of p38 knockdown on quercetin-induced ABCA1 mRNA (D) and protein (E) expression was examined by quantitative real-time PCR and Western blot analysis, respectively. The normalized level of mRNA or protein from parental cells without quercetin treatment was given as 1. F: Cholesterol efflux from p38 knockdown cells to media was measured in the presence of 10 μg/ml apoAI. Cholesterol efflux was expressed as the percentage of radioactivity in the medium relative to the total radioactivity (medium and cells). Values are mean ± SEM (n = 3–6). * P

    Article Snippet: Materials SB203580 (p38 inhibitor) and 5Z-7-Oxozeaenol (TAK1 inhibitor) were obtained from Calbiochem (San Diego, CA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Infection, shRNA, Luciferase, Radioactivity

    The p38 MARK signaling pathway is involved in pSTAT1-S727 elevation in PRRSV-infected cells. A. SB203580 treatment leads to inhibition of PRRSV-induced pSTAT1 elevation. MARC-145 cells were infected with PRRSV and treated with SB203580. At 24 hpi, the cells were harvested for Western blotting with antibodies against pSTAT1-S727, STAT1, and tubulin. B. PRRSV yield remains unchanged in the presence of SB203580. Cell culture supernatant samples of PRRSV-infected MARC-145 cells in the presence or absence of SB203580 were titrated. The viral yield is shown as log10 TCID50/ml. C. Cell viability assay of SB203580-treated MARC-145 cells. The cells were subjected for the assay 24 h after treatment. Relative percentages in comparison with mock-treated cells are shown.

    Journal: PLoS ONE

    Article Title: Induction of STAT1 Phosphorylation at Serine 727 and Expression of Proinflammatory Cytokines by Porcine Reproductive and Respiratory Syndrome Virus

    doi: 10.1371/journal.pone.0061967

    Figure Lengend Snippet: The p38 MARK signaling pathway is involved in pSTAT1-S727 elevation in PRRSV-infected cells. A. SB203580 treatment leads to inhibition of PRRSV-induced pSTAT1 elevation. MARC-145 cells were infected with PRRSV and treated with SB203580. At 24 hpi, the cells were harvested for Western blotting with antibodies against pSTAT1-S727, STAT1, and tubulin. B. PRRSV yield remains unchanged in the presence of SB203580. Cell culture supernatant samples of PRRSV-infected MARC-145 cells in the presence or absence of SB203580 were titrated. The viral yield is shown as log10 TCID50/ml. C. Cell viability assay of SB203580-treated MARC-145 cells. The cells were subjected for the assay 24 h after treatment. Relative percentages in comparison with mock-treated cells are shown.

    Article Snippet: Chemical SB203580 and MTA (Sigma, St. Louis, MO) were used at a final concentration of 20 µM and 1 mM, respectively, to treat cells for the length of time as indicated.

    Techniques: Infection, Inhibition, Western Blot, Cell Culture, Viability Assay

    VR-2385 infection leads to higher expression of proinflammatory cytokine genes than MLV and SB203580 treatment reduces their expression. MARC-145 cells were infected with VR-2385 or MLV and treated with SB203580. Mock-treated cells were included for control. The cells were harvested at 48 hpi for RNA isolation and RT-qPCR. Relative folds of transcript levels of IL-1β, IL-8, and ISG54 in comparison with mock-treated PRRSV-negative cells are shown. Error bars represent variation of three repeated experiments. Significant differences between paired samples are shown by “*” and “**”, which indicate P

    Journal: PLoS ONE

    Article Title: Induction of STAT1 Phosphorylation at Serine 727 and Expression of Proinflammatory Cytokines by Porcine Reproductive and Respiratory Syndrome Virus

    doi: 10.1371/journal.pone.0061967

    Figure Lengend Snippet: VR-2385 infection leads to higher expression of proinflammatory cytokine genes than MLV and SB203580 treatment reduces their expression. MARC-145 cells were infected with VR-2385 or MLV and treated with SB203580. Mock-treated cells were included for control. The cells were harvested at 48 hpi for RNA isolation and RT-qPCR. Relative folds of transcript levels of IL-1β, IL-8, and ISG54 in comparison with mock-treated PRRSV-negative cells are shown. Error bars represent variation of three repeated experiments. Significant differences between paired samples are shown by “*” and “**”, which indicate P

    Article Snippet: Chemical SB203580 and MTA (Sigma, St. Louis, MO) were used at a final concentration of 20 µM and 1 mM, respectively, to treat cells for the length of time as indicated.

    Techniques: Infection, Expressing, Isolation, Quantitative RT-PCR

    PRRSV infection of PAM cells leads to elevation of pSTAT1-S727. A. Elevation of pSTAT1-S727 in VR-2385-infected PAM cells detected by Western blotting. The cells were infected with VR-2385 and treated with SB203580. Mock-infected and mock-treated cells were included as controls. The blotting was done with antibodies against pSTAT1-S727, STAT1 and tubulin. Relative levels of pSTAT1-S727 compared to the mock-treated control are shown as folds below the images. B. PRRSV viral RNA copies in PAMs detected by RT-qPCR and shown as log10 per 10 ng total RNA. C D. Increased expression of IL-1β, CXCL10, IL-8, IL-10, and CCL2 in PRRSV-infected PAMs detected by RT-qPCR. The cells were infected with VR-2385 and treated with SB203580. Relative levels of gene expression are shown as folds in comparison with the mock-treated PRRSV-negative cells.

    Journal: PLoS ONE

    Article Title: Induction of STAT1 Phosphorylation at Serine 727 and Expression of Proinflammatory Cytokines by Porcine Reproductive and Respiratory Syndrome Virus

    doi: 10.1371/journal.pone.0061967

    Figure Lengend Snippet: PRRSV infection of PAM cells leads to elevation of pSTAT1-S727. A. Elevation of pSTAT1-S727 in VR-2385-infected PAM cells detected by Western blotting. The cells were infected with VR-2385 and treated with SB203580. Mock-infected and mock-treated cells were included as controls. The blotting was done with antibodies against pSTAT1-S727, STAT1 and tubulin. Relative levels of pSTAT1-S727 compared to the mock-treated control are shown as folds below the images. B. PRRSV viral RNA copies in PAMs detected by RT-qPCR and shown as log10 per 10 ng total RNA. C D. Increased expression of IL-1β, CXCL10, IL-8, IL-10, and CCL2 in PRRSV-infected PAMs detected by RT-qPCR. The cells were infected with VR-2385 and treated with SB203580. Relative levels of gene expression are shown as folds in comparison with the mock-treated PRRSV-negative cells.

    Article Snippet: Chemical SB203580 and MTA (Sigma, St. Louis, MO) were used at a final concentration of 20 µM and 1 mM, respectively, to treat cells for the length of time as indicated.

    Techniques: Infection, Western Blot, Quantitative RT-PCR, Expressing

    LBP gene overexpression enhanced the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65. LBP gene silencing inhibited the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65. SB203580 and SC-514 inhibited the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65, respectively, in A549 cells. Coimmunoprecipitation and western blotting were used to detect the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65. Phospho-p38 MAPK/LBP and phospho-p65/LBP relative densities were calculated to measure the strength of protein interactions. The result shows that LBP interacted with phospho-p38 MAPK and phospho-p65 weakly in the control group of cells ( p

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: LBP gene overexpression enhanced the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65. LBP gene silencing inhibited the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65. SB203580 and SC-514 inhibited the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65, respectively, in A549 cells. Coimmunoprecipitation and western blotting were used to detect the interaction between LBP and phospho-p38 MAPK and LBP and phospho-p65. Phospho-p38 MAPK/LBP and phospho-p65/LBP relative densities were calculated to measure the strength of protein interactions. The result shows that LBP interacted with phospho-p38 MAPK and phospho-p65 weakly in the control group of cells ( p

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Over Expression, Western Blot

    Expression of LBP and FKN mRNA in ARDS rat lung homogenates. The lung tissue was homogenized by sonicating in RIPA buffer. Total RNA of lung homogenates was isolated using chloroform, and the protocol of TRIzol kit was used according to the manufacturer's instructions. RT-PCR was used to analyze the LBP and FKN mRNA expression in ARDS rat lung tissues. The results suggest that LBPK95A, SB203580, and SC-514 suppressed the LPS-induced FKN reduction. ∗ represents p

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: Expression of LBP and FKN mRNA in ARDS rat lung homogenates. The lung tissue was homogenized by sonicating in RIPA buffer. Total RNA of lung homogenates was isolated using chloroform, and the protocol of TRIzol kit was used according to the manufacturer's instructions. RT-PCR was used to analyze the LBP and FKN mRNA expression in ARDS rat lung tissues. The results suggest that LBPK95A, SB203580, and SC-514 suppressed the LPS-induced FKN reduction. ∗ represents p

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    The expressions of phospho-p38 MAPK and phospho-p65 in A549 cells with and without plasmid transfection or inhibitors following LPS treatment. Western blotting was used to image the expression of phospho-p38 MAPK and phospho-p65 in A549 cells with and without plasmid transfection or inhibitors following LPS treatment. A549 cells of the LPS+LBP and LPS+LBP(−) groups were transfected with LBP plasmid DNA or LBPshRNA-expressing plasmid DNA, respectively, for 48mins following LPS treatment. A549 cells of the LPS+SB and LPS+SC groups were pretreated with SB203580 or SC-514 for 60 mins following LPS treatment. The cells were collected with cell lysis buffer 1 h after LPS stimulation for western blotting. The figure shows that there is increased expression of phospho-p38 MAPK and phospho-p65 in A549 following LPS treatment. Transfection with LBP plasmid DNA increased the expression of phospho-p38 MAPK and phospho-p65. Transfection with LBP shRNA plasmid DNA decreased the expression of phospho-p38 MAPK and phospho-p65. Pretreatment with SB203580 reduced the expression of phospho-p38 MAPK, and pretreatment with SC-514 reduced the expression of phospho-p65 protein. The relative densities of phospho-p38 MAPK/ β -actin (a and c) and phospho-p65/ β -actin (b and d). ∗ represents p

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: The expressions of phospho-p38 MAPK and phospho-p65 in A549 cells with and without plasmid transfection or inhibitors following LPS treatment. Western blotting was used to image the expression of phospho-p38 MAPK and phospho-p65 in A549 cells with and without plasmid transfection or inhibitors following LPS treatment. A549 cells of the LPS+LBP and LPS+LBP(−) groups were transfected with LBP plasmid DNA or LBPshRNA-expressing plasmid DNA, respectively, for 48mins following LPS treatment. A549 cells of the LPS+SB and LPS+SC groups were pretreated with SB203580 or SC-514 for 60 mins following LPS treatment. The cells were collected with cell lysis buffer 1 h after LPS stimulation for western blotting. The figure shows that there is increased expression of phospho-p38 MAPK and phospho-p65 in A549 following LPS treatment. Transfection with LBP plasmid DNA increased the expression of phospho-p38 MAPK and phospho-p65. Transfection with LBP shRNA plasmid DNA decreased the expression of phospho-p38 MAPK and phospho-p65. Pretreatment with SB203580 reduced the expression of phospho-p38 MAPK, and pretreatment with SC-514 reduced the expression of phospho-p65 protein. The relative densities of phospho-p38 MAPK/ β -actin (a and c) and phospho-p65/ β -actin (b and d). ∗ represents p

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Plasmid Preparation, Transfection, Western Blot, Expressing, Lysis, shRNA

    Expression of phospho-p38 MAPK and phospho-p65 in lung tissue homogenates from rats with ARDS. Western blotting was used to detect the expression of phospho-p38 MAPK and phospho-p65 in the ARDS rat model with and without inhibitors following LPS treatment. LBPK95A, SB203580, and SC-514 inhibited the LPS-induced expression of phospho-p38 MAPK (a) and phospho-p65 (b) as seen from the relative densities of phospho-p38 MAPK/ β -actin (c) and phospho-p65/ β -actin (d). ∗ represents p

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: Expression of phospho-p38 MAPK and phospho-p65 in lung tissue homogenates from rats with ARDS. Western blotting was used to detect the expression of phospho-p38 MAPK and phospho-p65 in the ARDS rat model with and without inhibitors following LPS treatment. LBPK95A, SB203580, and SC-514 inhibited the LPS-induced expression of phospho-p38 MAPK (a) and phospho-p65 (b) as seen from the relative densities of phospho-p38 MAPK/ β -actin (c) and phospho-p65/ β -actin (d). ∗ represents p

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Expressing, Western Blot

    (a) LBP promoted phospho-p38 MAPK translocation, LBP shRNA, and SB203580 inhibited phospho-p38 MAPK translocation in response to LPS stimulation, and (b) LBP promoted phospho-p65 translocation, LBP shRNA, and SC-514 inhibited phospho-p65 translocation in response to LPS stimulation. Immunohistochemical staining was used to image the effect of LBP overexpression, LBP silencing, SB203580, and SC-514 on the activation of p38 MAPK and NF-B in A549 cells. The cells were fixed in 4% paraformaldehyde and permeabilized. Following incubation with anti-phospho-p38 MAPK or anti-phospho-p65 antibody overnight at 4°C, a fluorescent secondary antibody was used for nuclear staining. The cell slides were mounted with propidium iodide- (PI-) containing mounting media and visualized using a confocal laser scanning microscope. The photomicrographs are at 400x magnification.

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: (a) LBP promoted phospho-p38 MAPK translocation, LBP shRNA, and SB203580 inhibited phospho-p38 MAPK translocation in response to LPS stimulation, and (b) LBP promoted phospho-p65 translocation, LBP shRNA, and SC-514 inhibited phospho-p65 translocation in response to LPS stimulation. Immunohistochemical staining was used to image the effect of LBP overexpression, LBP silencing, SB203580, and SC-514 on the activation of p38 MAPK and NF-B in A549 cells. The cells were fixed in 4% paraformaldehyde and permeabilized. Following incubation with anti-phospho-p38 MAPK or anti-phospho-p65 antibody overnight at 4°C, a fluorescent secondary antibody was used for nuclear staining. The cell slides were mounted with propidium iodide- (PI-) containing mounting media and visualized using a confocal laser scanning microscope. The photomicrographs are at 400x magnification.

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Translocation Assay, shRNA, Immunohistochemistry, Staining, Over Expression, Activation Assay, Incubation, Laser-Scanning Microscopy

    LBP gene overexpression further decreased LPS-induced downregulation of FKN mRNA and protein expression; LBP gene silencing, SB203580, and SC-514 suppressed LPS-induced downregulation of FKN mRNA and protein expression, respectively, in A549 cells. RT-PCR and ELISA were used to analyze the LBP and FKN mRNA and protein expression, respectively, in A549 cells. The LBP mRNA expression is shown in (a). The relative LBP mRNA levels (normalized to GAPDH mRNA) in LPS-stimulated cells are significantly higher than those in control cells ( p

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: LBP gene overexpression further decreased LPS-induced downregulation of FKN mRNA and protein expression; LBP gene silencing, SB203580, and SC-514 suppressed LPS-induced downregulation of FKN mRNA and protein expression, respectively, in A549 cells. RT-PCR and ELISA were used to analyze the LBP and FKN mRNA and protein expression, respectively, in A549 cells. The LBP mRNA expression is shown in (a). The relative LBP mRNA levels (normalized to GAPDH mRNA) in LPS-stimulated cells are significantly higher than those in control cells ( p

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    SB203580 and SC-514 ameliorated LPS-induced lung injury and inflammation. Hematoxylin and eosin staining was used to determine the LPS-induced lung injury and inflammatory response in the lung tissue of the ARDS rat model. (a) The control group rats (A) had a distinct framework with complete alveolar walls and interstitium without exfiltration. The LPS group rats (B) showed edema, neutrophil infiltration, hemorrhage, moderate bronchiole epithelial desquamation, and minimal hyaline membrane formation. Histotological analysis also show that rats receiving LBPK95A, SB203508, or SC514 injection had substantially less inflammatory cell infiltration, edema, hemorrhage, and thickness of alveolar walls in comparison to rats treated with LPS ((C), (D), and (E), resp.). The photomicrographs are at 400x magnification. ((b) and (c)) The total number of cells, neutrophil number, and neutrophil ratio in BALF were higher in the LPS group than that in the control group and lower in the LPS+LBPK95A, LPS+SB, and LPS+SC groups than that in the LPS group ( p

    Journal: Mediators of Inflammation

    Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF-κB

    doi: 10.1155/2017/9734837

    Figure Lengend Snippet: SB203580 and SC-514 ameliorated LPS-induced lung injury and inflammation. Hematoxylin and eosin staining was used to determine the LPS-induced lung injury and inflammatory response in the lung tissue of the ARDS rat model. (a) The control group rats (A) had a distinct framework with complete alveolar walls and interstitium without exfiltration. The LPS group rats (B) showed edema, neutrophil infiltration, hemorrhage, moderate bronchiole epithelial desquamation, and minimal hyaline membrane formation. Histotological analysis also show that rats receiving LBPK95A, SB203508, or SC514 injection had substantially less inflammatory cell infiltration, edema, hemorrhage, and thickness of alveolar walls in comparison to rats treated with LPS ((C), (D), and (E), resp.). The photomicrographs are at 400x magnification. ((b) and (c)) The total number of cells, neutrophil number, and neutrophil ratio in BALF were higher in the LPS group than that in the control group and lower in the LPS+LBPK95A, LPS+SB, and LPS+SC groups than that in the LPS group ( p

    Article Snippet: Groupings The cells were divided into seven groups as follows: control group (CTL); LPS group (LPS, LPS treatment at 10 μ g/mL, L2880, Sigma-Aldrich, USA); LPS and LBP group (LPS+LBP(+), the cells were transfected with the LBP plasmid DNA (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine® 2000 transfection reagent (11668-027, Invitrogen, USA) and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and LBP(−) group (LPS+LBP(−), the cells were transfected with the pGCU6/Neo-LBP shRNA-expressing plasmid DNA (ShLBP) which targeted the LBP mRNA sequence (5′-GCATCAGCATTTCGGTCAACC-3′) (Genechem Co., Ltd., Shanghai, China) by using Lipofectamine 2000 transfection reagent and maintained in RPMI 1640 medium for 48 h following LPS treatment); LPS and SB203580 group (LPS+SB, the cells were preincubated with 10 μ M SB203580 (S8307, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SB203580 was as described previously [ ]); LPS and SC-514 group (LPS+SC, the cells were preincubated with 10 μ M SC-514 (SML0557, Sigma-Aldrich, USA) for 60 minutes following LPS treatment, and the concentration of SC-514 was as described previously); and tumor necrosis factor-α (TNF-α ) group (TNF-α , TNF-α treatment at 50 ng/mL for positive control (H8916, Sigma-Aldrich, USA), the concentration of TNF-α was as described previously [ ]).

    Techniques: Staining, Injection