p38 Cell Signaling Technology Inc Search Results


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  • 93
    Cell Signaling Technology Inc technology p38
    Inhibitory effects of elaidic acid on autophagy induction by saturated FAs. (A, B) U2OS cells stably expressing GFP-LC3 were treated with 500 μM FA, or 10 μM of the autophagy-inducer subset from the ENZO SCREEN-WELL autophagy library, in the presence or absence of 500 μM EL for 6 h. Autophagy was assessed after fixation by measuring the area of LC3 dots within each cell. The relative difference with and without EL co-treatment was calculated and is reported as barchart in A. Representative images are shown in B. Scale bar equals 10 μm. (C) U2OS cells were treated with either vector (ethanol 0.5%), 500 μM OL, PA, or stearate in the presence or absence of 500 μM elaidate for 6 h. Thereafter, LC3 lipidation and p62 degradation and <t>p38</t> activation were measured by immunoblot as indicators for autophagy. (D) U2OS cells treated as in C with OL, PA, and stearate in the presence or absence of elaidate, were harvested and analyzed with GC/MS to measure the intracellular FA concentration and to test the effect of EL on FA uptake into cells. Data are means ± SEM of at least three independent experiments (*=p
    Technology P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1563 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 mapk
    A CaMKII inhibitor, KN93, suppresses GW7845-induced <t>MAPK</t> activation. BU-11 cells were pretreated with Vh (DMSO, 0.1%) or KN93 (1–5μM) for 30 min and then treated with Vh (ethanol:DMSO, 50:50, 0.1%) or GW7845 (40μM) for 30 min. Cytoplasmic extracts were prepared and analyzed for <t>p38</t> MAPK activation by detection of phosphorylated p38 MAPK by immunoblotting (A), for JNK activation by in vitro kinase assay (B), and for endogenous p38 MAPK/JNK activity by detection of phosphorylated ATF-2 by immunoblotting (C). Protein expression was quantified as described in the Materials and Methods and presented as “Fold Change from Naive.” Data are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p
    Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 2694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38
    <t>p38</t> and JNK phosphorylation in the CPu in D1, D3 receptor mutant, and wild-type mice is not obviously changed after acute cocaine treatment. Time course of cocaine-induced phosphorylation of p38 and JNK in the CPu in wild-type ( A ), D1 ( B ), and D3 receptor mutant ( C ) mice. The phosphorylation status of p38 and JNK was determined 10, 20, 60, and 120 min after a cocaine injection (30 mg/kg, i.p.). Total cell extracts were analyzed by Western blotting using antibodies against dually phosphorylated-p38 (Thr 180 and Tyr 182 ), dually phosphorylated-JNK (Thr 183 and Tyr 185 ), total p38, or JNK, respectively. The same set of mice used in probing ERK activation was used in analyzing p38 and JNK activation after cocaine injections.
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    Cell Signaling Technology Inc phospoho p38
    <t>p38</t> and JNK phosphorylation in the CPu in D1, D3 receptor mutant, and wild-type mice is not obviously changed after acute cocaine treatment. Time course of cocaine-induced phosphorylation of p38 and JNK in the CPu in wild-type ( A ), D1 ( B ), and D3 receptor mutant ( C ) mice. The phosphorylation status of p38 and JNK was determined 10, 20, 60, and 120 min after a cocaine injection (30 mg/kg, i.p.). Total cell extracts were analyzed by Western blotting using antibodies against dually phosphorylated-p38 (Thr 180 and Tyr 182 ), dually phosphorylated-JNK (Thr 183 and Tyr 185 ), total p38, or JNK, respectively. The same set of mice used in probing ERK activation was used in analyzing p38 and JNK activation after cocaine injections.
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    Cell Signaling Technology Inc p38 mapk assay kits
    Effect of benidipine on ERK1/2 and <t>p38</t> <t>MAPK</t> activity in ischemic-reperfused myocardial tissue. The ERK1/2 and p38 MAP kinase activity assays were performed by using ERK1/2 or p38 MAPK assay kits. EIk-1 fusion protein or ATF-2 fusion protein was used as substrate for ERK1/2 or p38 MAPK, respectively. # P
    P38 Mapk Assay Kits, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 p p38 proteins
    Effect of benidipine on ERK1/2 and <t>p38</t> <t>MAPK</t> activity in ischemic-reperfused myocardial tissue. The ERK1/2 and p38 MAP kinase activity assays were performed by using ERK1/2 or p38 MAPK assay kits. EIk-1 fusion protein or ATF-2 fusion protein was used as substrate for ERK1/2 or p38 MAPK, respectively. # P
    Phospho P38 P P38 Proteins, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 p p38 standards
    Effect of benidipine on ERK1/2 and <t>p38</t> <t>MAPK</t> activity in ischemic-reperfused myocardial tissue. The ERK1/2 and p38 MAP kinase activity assays were performed by using ERK1/2 or p38 MAPK assay kits. EIk-1 fusion protein or ATF-2 fusion protein was used as substrate for ERK1/2 or p38 MAPK, respectively. # P
    Phospho P38 P P38 Standards, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc sirna against p38
    IL-1β upregulates MMP2 and MMP9 expression and activity by activating <t>p38.</t> A : RT-PCR analysis showed that MMP2 and MMP9 mRNA were upregulated in both AGS and MKN-45 cells in response to treatment with IL-1β; this effect was blocked by p38 <t>siRNA</t> or the p38 inhibitor SB202190. B : Quantification of the expression of MMP2 and MMP9 mRNA normalized to GAPDH mRNA. ** P
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    Cell Signaling Technology Inc p38 mitogen activated protein kinases p38
    PD-mediated phosphorylation of proinflammatory <t>p38</t> and JNK1/2 is reduced by chemical or genetic ablation of sEH. Phosphorylation and activation of p38 and JNK1/2 were quantified from all groups by normalizing band intensity to that of α -tubulin. (A) Original blots displaying two randomly selected animals. (B and C). Bar graphs of phosphorylation status of p38 and JNK1/2. Mean band intensity is measured for all six mice and is represented as arbitrary units (mean ± S.E.M.) (* P
    P38 Mitogen Activated Protein Kinases P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies against p p38 p38 arid1a
    PD-mediated phosphorylation of proinflammatory <t>p38</t> and JNK1/2 is reduced by chemical or genetic ablation of sEH. Phosphorylation and activation of p38 and JNK1/2 were quantified from all groups by normalizing band intensity to that of α -tubulin. (A) Original blots displaying two randomly selected animals. (B and C). Bar graphs of phosphorylation status of p38 and JNK1/2. Mean band intensity is measured for all six mice and is represented as arbitrary units (mean ± S.E.M.) (* P
    Antibodies Against P P38 P38 Arid1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 pp38 4631 cell signaling
    PD-mediated phosphorylation of proinflammatory <t>p38</t> and JNK1/2 is reduced by chemical or genetic ablation of sEH. Phosphorylation and activation of p38 and JNK1/2 were quantified from all groups by normalizing band intensity to that of α -tubulin. (A) Original blots displaying two randomly selected animals. (B and C). Bar graphs of phosphorylation status of p38 and JNK1/2. Mean band intensity is measured for all six mice and is represented as arbitrary units (mean ± S.E.M.) (* P
    Phospho P38 Pp38 4631 Cell Signaling, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p38 inhibitor sb203580
    ERK and <t>p38</t> play opposite roles in regulating monocyte migration induced by LPS and MCP-1. A. WT monocytes were pre-treated with p38 inhibitor <t>SB203580</t> (SB; 10 μM), ERK inhibitor PD98059 (PD; 50) or JNK inhibitor SP600125 (SP; 10 μM) for
    P38 Inhibitor Sb203580, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosoh p38 p p38
    ERK and <t>p38</t> play opposite roles in regulating monocyte migration induced by LPS and MCP-1. A. WT monocytes were pre-treated with p38 inhibitor <t>SB203580</t> (SB; 10 μM), ERK inhibitor PD98059 (PD; 50) or JNK inhibitor SP600125 (SP; 10 μM) for
    Phosoh P38 P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p38 pthr180 tyr182
    ERK and <t>p38</t> play opposite roles in regulating monocyte migration induced by LPS and MCP-1. A. WT monocytes were pre-treated with p38 inhibitor <t>SB203580</t> (SB; 10 μM), ERK inhibitor PD98059 (PD; 50) or JNK inhibitor SP600125 (SP; 10 μM) for
    P38 Pthr180 Tyr182, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphoplus p38 mapk antibody
    Keratinocyte ligation to fibrillar type I collagen induced phosphorylation of ERK and <t>p38</t> <t>MAPK</t> but not JNK Primary keratinocytes were plated on fibrillar type I collagen, and cell lysates were harvested at the designated time points. The 0-min cells were keratinocytes sampled before plating on collagen. Phosphorylated (p-) and total ERK, p38, and JNK were visualized by immunoblot using specific antibodies. Results are representative of three independent experiments with keratinocytes from 3 different batches of cells.
    Phosphoplus P38 Mapk Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 map kinasethr180 tyr182 p p38
    Keratinocyte ligation to fibrillar type I collagen induced phosphorylation of ERK and <t>p38</t> <t>MAPK</t> but not JNK Primary keratinocytes were plated on fibrillar type I collagen, and cell lysates were harvested at the designated time points. The 0-min cells were keratinocytes sampled before plating on collagen. Phosphorylated (p-) and total ERK, p38, and JNK were visualized by immunoblot using specific antibodies. Results are representative of three independent experiments with keratinocytes from 3 different batches of cells.
    Phospho P38 Map Kinasethr180 Tyr182 P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti p38 p p38
    Keratinocyte ligation to fibrillar type I collagen induced phosphorylation of ERK and <t>p38</t> <t>MAPK</t> but not JNK Primary keratinocytes were plated on fibrillar type I collagen, and cell lysates were harvested at the designated time points. The 0-min cells were keratinocytes sampled before plating on collagen. Phosphorylated (p-) and total ERK, p38, and JNK were visualized by immunoblot using specific antibodies. Results are representative of three independent experiments with keratinocytes from 3 different batches of cells.
    Rabbit Polyclonal Anti P38 P P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibitory effects of elaidic acid on autophagy induction by saturated FAs. (A, B) U2OS cells stably expressing GFP-LC3 were treated with 500 μM FA, or 10 μM of the autophagy-inducer subset from the ENZO SCREEN-WELL autophagy library, in the presence or absence of 500 μM EL for 6 h. Autophagy was assessed after fixation by measuring the area of LC3 dots within each cell. The relative difference with and without EL co-treatment was calculated and is reported as barchart in A. Representative images are shown in B. Scale bar equals 10 μm. (C) U2OS cells were treated with either vector (ethanol 0.5%), 500 μM OL, PA, or stearate in the presence or absence of 500 μM elaidate for 6 h. Thereafter, LC3 lipidation and p62 degradation and p38 activation were measured by immunoblot as indicators for autophagy. (D) U2OS cells treated as in C with OL, PA, and stearate in the presence or absence of elaidate, were harvested and analyzed with GC/MS to measure the intracellular FA concentration and to test the effect of EL on FA uptake into cells. Data are means ± SEM of at least three independent experiments (*=p

    Journal: EBioMedicine

    Article Title: Trans-Fats Inhibit Autophagy Induced by Saturated Fatty Acids

    doi: 10.1016/j.ebiom.2018.03.028

    Figure Lengend Snippet: Inhibitory effects of elaidic acid on autophagy induction by saturated FAs. (A, B) U2OS cells stably expressing GFP-LC3 were treated with 500 μM FA, or 10 μM of the autophagy-inducer subset from the ENZO SCREEN-WELL autophagy library, in the presence or absence of 500 μM EL for 6 h. Autophagy was assessed after fixation by measuring the area of LC3 dots within each cell. The relative difference with and without EL co-treatment was calculated and is reported as barchart in A. Representative images are shown in B. Scale bar equals 10 μm. (C) U2OS cells were treated with either vector (ethanol 0.5%), 500 μM OL, PA, or stearate in the presence or absence of 500 μM elaidate for 6 h. Thereafter, LC3 lipidation and p62 degradation and p38 activation were measured by immunoblot as indicators for autophagy. (D) U2OS cells treated as in C with OL, PA, and stearate in the presence or absence of elaidate, were harvested and analyzed with GC/MS to measure the intracellular FA concentration and to test the effect of EL on FA uptake into cells. Data are means ± SEM of at least three independent experiments (*=p

    Article Snippet: Membranes were probed overnight at 4 °C with primary antibodies specific for LC3 (#2775, Cell Signaling Technology), p62 (ab56416, Abcam), Atg5 (A2859, Sigma-Aldrich), GAPDH (ab8254, Abcam), XBP1s (BLE619502, Biolegend, San Diego, CA, USA), P-p38 (#9211, Cell Signaling Technology), p38 (#9212, Cell Signaling Technology), followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech, Birmingham, AL, USA).

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Activation Assay, Gas Chromatography-Mass Spectrometry, Concentration Assay

    Involvement of the MAPK and PI3K/Akt pathways in the methamphetamine-induced translocation of STAT3 into the nucleus. ( A – C ) Representative western blot showing the effects of methamphetamine (150 μM) on the expression of STAT in whole-cell lysates ( A ), nucleus ( B ) and cytoplasm ( C ) of BV-2 cells. ( D ) Representative western blot showing the effects of the STAT inhibitor stattic on the methamphetamine-induced the expression of iNOS, arginase and SOCS3 in BV-2 cells. ( E ) Densitometric analyses of five separate experiments suggest that Meth induced the ratio of iNOS and arginase expression, which was attenuated by stattic pretreatment. ( F ) Representative western blot showing that pretreatment with the ERK inhibitor (U0126, 10 μM), p38 inhibitor (SP600125, 10 μM), JNK inhibitor (SB203580, 10 μM) and Akt inhibitor (LY294002, 10 μM) affected the expression of STAT3 in BV-2 cells. ( G ) Densitometric analyses suggesting that methamphetamine induced STAT3 expression, which was attenuated by pretreatment with SP600125, SB203580 and LY294002 but not U0126. *p

    Journal: Scientific Reports

    Article Title: Molecular mechanisms underlying the involvement of the sigma-1 receptor in methamphetamine-mediated microglial polarization

    doi: 10.1038/s41598-017-11065-8

    Figure Lengend Snippet: Involvement of the MAPK and PI3K/Akt pathways in the methamphetamine-induced translocation of STAT3 into the nucleus. ( A – C ) Representative western blot showing the effects of methamphetamine (150 μM) on the expression of STAT in whole-cell lysates ( A ), nucleus ( B ) and cytoplasm ( C ) of BV-2 cells. ( D ) Representative western blot showing the effects of the STAT inhibitor stattic on the methamphetamine-induced the expression of iNOS, arginase and SOCS3 in BV-2 cells. ( E ) Densitometric analyses of five separate experiments suggest that Meth induced the ratio of iNOS and arginase expression, which was attenuated by stattic pretreatment. ( F ) Representative western blot showing that pretreatment with the ERK inhibitor (U0126, 10 μM), p38 inhibitor (SP600125, 10 μM), JNK inhibitor (SB203580, 10 μM) and Akt inhibitor (LY294002, 10 μM) affected the expression of STAT3 in BV-2 cells. ( G ) Densitometric analyses suggesting that methamphetamine induced STAT3 expression, which was attenuated by pretreatment with SP600125, SB203580 and LY294002 but not U0126. *p

    Article Snippet: The membranes were then incubated with primary antibodies for p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-Akt/Akt, suppressor of cytokine signaling 3 (SOCS3) (Cell Signaling, 1:1000), signal transducer and activator of transcription 3 (STAT3) (Proteintech, 1:1000), iNOS (Abcam, 1:1000) and β-actin (Santa Cruz, 1:500) as previously described , .

    Techniques: Translocation Assay, Western Blot, Expressing

    Methamphetamine induced the activation of the MAPK and PI3K/Akt pathways. ( A ) Representative western blot showing the effects of methamphetamine (150 μM) on the phosphorylation of ERK, JNK, p38 and Akt in BV-2 cells. ( B ) Western blot and densitometric analyses showing that pretreatment with the sigma-1 receptor antagonist BD1047 attenuated the methamphetamine-induced phosphorylation of ERK, JNK, p38 and Akt in BV-2 cells. ( C ) Representative western blot and densitometric analyses showing the effects of apocynin on the methamphetamine-induced phosphorylation of ERK, JNK, p38 and Akt in BV-2 cells. *p

    Journal: Scientific Reports

    Article Title: Molecular mechanisms underlying the involvement of the sigma-1 receptor in methamphetamine-mediated microglial polarization

    doi: 10.1038/s41598-017-11065-8

    Figure Lengend Snippet: Methamphetamine induced the activation of the MAPK and PI3K/Akt pathways. ( A ) Representative western blot showing the effects of methamphetamine (150 μM) on the phosphorylation of ERK, JNK, p38 and Akt in BV-2 cells. ( B ) Western blot and densitometric analyses showing that pretreatment with the sigma-1 receptor antagonist BD1047 attenuated the methamphetamine-induced phosphorylation of ERK, JNK, p38 and Akt in BV-2 cells. ( C ) Representative western blot and densitometric analyses showing the effects of apocynin on the methamphetamine-induced phosphorylation of ERK, JNK, p38 and Akt in BV-2 cells. *p

    Article Snippet: The membranes were then incubated with primary antibodies for p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-Akt/Akt, suppressor of cytokine signaling 3 (SOCS3) (Cell Signaling, 1:1000), signal transducer and activator of transcription 3 (STAT3) (Proteintech, 1:1000), iNOS (Abcam, 1:1000) and β-actin (Santa Cruz, 1:500) as previously described , .

    Techniques: Activation Assay, Western Blot

    Tat-mediated induction of ICAM-1 expression involves MAPK signaling pathways. ( A ) Western blot analysis demonstrated time-dependent activation of ERK, JNK and p38 by Tat in HUVECs. ( B ) Inhibition of the ERK, JNK and p38 MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) inhibitors resulted in amelioration of Tat-mediated induction of ICAM-1 expression. ( C ) Pharmacological inhibition of MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) resulted in amelioration of Tat-mediated induction of monocyte adhesion. All the data are presented as mean ± SD of three independent experiments. **p

    Journal: PLoS ONE

    Article Title: HIV Tat Induces Expression of ICAM-1 in HUVECs: Implications for miR-221/-222 in HIV-Associated Cardiomyopathy

    doi: 10.1371/journal.pone.0060170

    Figure Lengend Snippet: Tat-mediated induction of ICAM-1 expression involves MAPK signaling pathways. ( A ) Western blot analysis demonstrated time-dependent activation of ERK, JNK and p38 by Tat in HUVECs. ( B ) Inhibition of the ERK, JNK and p38 MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) inhibitors resulted in amelioration of Tat-mediated induction of ICAM-1 expression. ( C ) Pharmacological inhibition of MAPK pathways by MEK1/2 (U0126, 20 µM), JNK (SP600125, 20 µM) and p38 (SB203580, 20 µM) resulted in amelioration of Tat-mediated induction of monocyte adhesion. All the data are presented as mean ± SD of three independent experiments. **p

    Article Snippet: Western blots were then probed with antibodies recognizing the ICAM-1, ERK1/2, JNK, p38,NF-κB p65 (1∶200; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1∶4000; Sigma, St. Louis, MO, USA).

    Techniques: Expressing, Western Blot, Activation Assay, Inhibition

    Bone marrow and spleen from Gata1 low mice contain levels greater than normal both of signaling elements downstream to TPO and of those downstream to TGF-β. ( a , b ) Western blot determinations of the JAK2, STAT5 and pSTAT5 content of bone marrow and spleen from wild-type and Gata1 low mice (each lane). GAPDH was used as loading control. Quantification was obtained by normalizing each band to the corresponding GAPDH level and is presented as mean (±s.d.) with three mice per experimental group. P -values were determined by analysis of variance (ANOVA). ( c , d ) Western blot determinations of the SMAD2/3, phosphorylated p38 (p-p38), p38 and EZH2 content in the bone marrow and spleen from wild-type and Gata1 low mice (each line a different mouse). GAPDH was used as a loading control. Quantification was obtained by normalizing p-p38 toward the corresponding p38 band, and SMAD2/3, p38 and EZH2 signals against GAPDH. P -values were calculated by ANOVA. EZH2, enhancer of zeste homolog 2.

    Journal: Blood Cancer Journal

    Article Title: The thrombopoietin/MPL axis is activated in the Gata1low mouse model of myelofibrosis and is associated with a defective RPS14 signature

    doi: 10.1038/bcj.2017.51

    Figure Lengend Snippet: Bone marrow and spleen from Gata1 low mice contain levels greater than normal both of signaling elements downstream to TPO and of those downstream to TGF-β. ( a , b ) Western blot determinations of the JAK2, STAT5 and pSTAT5 content of bone marrow and spleen from wild-type and Gata1 low mice (each lane). GAPDH was used as loading control. Quantification was obtained by normalizing each band to the corresponding GAPDH level and is presented as mean (±s.d.) with three mice per experimental group. P -values were determined by analysis of variance (ANOVA). ( c , d ) Western blot determinations of the SMAD2/3, phosphorylated p38 (p-p38), p38 and EZH2 content in the bone marrow and spleen from wild-type and Gata1 low mice (each line a different mouse). GAPDH was used as a loading control. Quantification was obtained by normalizing p-p38 toward the corresponding p38 band, and SMAD2/3, p38 and EZH2 signals against GAPDH. P -values were calculated by ANOVA. EZH2, enhancer of zeste homolog 2.

    Article Snippet: Western blot analysis Protein extracts separated on SDS-polyacrylamide gel electrophoresis, were blotted on nitrocellulose membranes and probed with antibodies against JAK2 (D2E12 #3230), pSTAT5 (#9351), Ezh2 (Ac22 #3147), SMAD2/3 (#5339), p38 (#9213) and p-p38 (T180/Y182 #4511) (all from Cell Signaling, Boston, MA, USA), STAT5 (#sc-835, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (#CB1001, Calbiochem, San Diego, CA, USA).

    Techniques: Mouse Assay, Western Blot

    Involvement of the MAPK, NF-κB and STAT3 pathways in the ACE2/Ang-(1–7)/Mas axis mediated protection in EAU eye. (a) The phosphorylation levels of p38 MAPK, ERK1/2, JNK, IκB-α and STAT3 (p-p38 MAPK, p-ERK1/2, p-JNK, p-IκB-α and p-STAT3) in the AAV8-ACE2+EAU group compared with the AAV8-eGFP+EAU and EAU groups were determined by Western blotting (* p

    Journal: Scientific Reports

    Article Title: AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-κB and STAT3 Pathways

    doi: 10.1038/srep31912

    Figure Lengend Snippet: Involvement of the MAPK, NF-κB and STAT3 pathways in the ACE2/Ang-(1–7)/Mas axis mediated protection in EAU eye. (a) The phosphorylation levels of p38 MAPK, ERK1/2, JNK, IκB-α and STAT3 (p-p38 MAPK, p-ERK1/2, p-JNK, p-IκB-α and p-STAT3) in the AAV8-ACE2+EAU group compared with the AAV8-eGFP+EAU and EAU groups were determined by Western blotting (* p

    Article Snippet: Membranes were blocked with 5% skim milk or 5% bovine serum albumin (BSA) and incubated with specific primary antibodies against mouse ACE2 (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), p-IκBα (1:50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-actin(1:100, ABCAM, Cambridge, MA, USA), p38, JNK, ERK1/2, phosphorylated p38 (p-p38), p-ERK1/2 (1:1000, Cell Signaling Technology, Danvers, MA, USA), JNK, p-JNK (1:500, Cell Signaling Technology, Danvers, MA, USA), STAT3, p-STAT3 (Tyr705) (1:1000, Cell Signaling Technology, Danvers, MA, USA) over night at 4 °C, followed by the secondary antibody (1:3000, ABCAM, Cambridge, MA, USA) at 37 °C for 1 hour.

    Techniques: Western Blot

    The expression of IFN-γ and IL-17 in response to IRBP stimulation with or without MAPK, NF-κB or STAT3 inhibitors. Lymphocytes were extracted from the spleens and draining lymph nodes of EAU mice. The lymphocytes were pre-treated with or without the MAPK, NF-κB or STAT3 inhibitors for 30 minutes and then incubated with IRBP for 72 hours. The protein expression levels of IFN-γ and IL-17 were measured by ELISA 72 hours after stimulation by IRBP with or without the presence of the p38 MAPK inhibitor (SB203580, SB), JNK inhibitor (SP600125, SP), ERK1/2 inhibitor (PD98059, PD), NF-κB inhibitor (BAY11-7082, BAY) at a final concentration of 10 μM and STAT3 inhibitor S3I-201 at the concentration of 100 μM. The data were shown as mean ± SEM (*** p

    Journal: Scientific Reports

    Article Title: AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-κB and STAT3 Pathways

    doi: 10.1038/srep31912

    Figure Lengend Snippet: The expression of IFN-γ and IL-17 in response to IRBP stimulation with or without MAPK, NF-κB or STAT3 inhibitors. Lymphocytes were extracted from the spleens and draining lymph nodes of EAU mice. The lymphocytes were pre-treated with or without the MAPK, NF-κB or STAT3 inhibitors for 30 minutes and then incubated with IRBP for 72 hours. The protein expression levels of IFN-γ and IL-17 were measured by ELISA 72 hours after stimulation by IRBP with or without the presence of the p38 MAPK inhibitor (SB203580, SB), JNK inhibitor (SP600125, SP), ERK1/2 inhibitor (PD98059, PD), NF-κB inhibitor (BAY11-7082, BAY) at a final concentration of 10 μM and STAT3 inhibitor S3I-201 at the concentration of 100 μM. The data were shown as mean ± SEM (*** p

    Article Snippet: Membranes were blocked with 5% skim milk or 5% bovine serum albumin (BSA) and incubated with specific primary antibodies against mouse ACE2 (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), p-IκBα (1:50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-actin(1:100, ABCAM, Cambridge, MA, USA), p38, JNK, ERK1/2, phosphorylated p38 (p-p38), p-ERK1/2 (1:1000, Cell Signaling Technology, Danvers, MA, USA), JNK, p-JNK (1:500, Cell Signaling Technology, Danvers, MA, USA), STAT3, p-STAT3 (Tyr705) (1:1000, Cell Signaling Technology, Danvers, MA, USA) over night at 4 °C, followed by the secondary antibody (1:3000, ABCAM, Cambridge, MA, USA) at 37 °C for 1 hour.

    Techniques: Expressing, Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Effects of ATF3 on the ERK and JNK signaling pathways. (A and B) The levels of total and phosphorylated MEK1/2, ERK1/2, JNK, P38, P90RSK and AKT in the heart tissues of mice in the indicated groups (n = 6). A, Representative blots. B, Quantitative results. * P

    Journal: PLoS ONE

    Article Title: Activating Transcription Factor 3 Deficiency Promotes Cardiac Hypertrophy, Dysfunction, and Fibrosis Induced by Pressure Overload

    doi: 10.1371/journal.pone.0026744

    Figure Lengend Snippet: Effects of ATF3 on the ERK and JNK signaling pathways. (A and B) The levels of total and phosphorylated MEK1/2, ERK1/2, JNK, P38, P90RSK and AKT in the heart tissues of mice in the indicated groups (n = 6). A, Representative blots. B, Quantitative results. * P

    Article Snippet: The primary antibodies included antibodies specific for p-MEK1/2 (Cell Signaling Technology, 9154), T-MEK1/2 (Cell Signaling Technology, 9122), p-ERK1/2 (Cell Signaling Technology, 4370), T-ERK1/2 (Cell Signaling Technology, 4695), p-P38 (Cell Signaling Technology, 4511), T-P38 (Cell Signaling Technology, 9212), p-JNK (Cell Signaling Technology, 4668), T-JNK (Cell Signaling Technology, 9258), p-p90RSK(Cell Signaling Technology, 9335), T-p90RSK (Cell Signaling Technology, 9347), p-AKT (Cell Signaling Technology, 4060), T-AKT (Cell Signaling Technology, 4691), GAPDH (Cell Signaling Technology, 2118), and ATF3 (Santa Cruz Biotechnology, sc-188).

    Techniques: Mouse Assay

    Inhibitory effect of PVE on MAPKs (A–D) and AP-1 (E–G) . NHDFs were irradiated with UVB (144 mJ/cm 2 ) followed by treatment with PVE (1, 10, 100 μg/mL) for 1.5 hours. Phosphorylation and nonphosphorylation of JNK, ERK, p38, c-Fos, and c-Jun were measured. The band intensities were quantified by densitometry, normalized to the level of β-actin, and calculated as the percentage of the basal response. Values are mean ± SD. ## p

    Journal: Rejuvenation Research

    Article Title: Prunella vulgaris L. Exerts a Protective Effect Against Extrinsic Aging Through NF-κB, MAPKs, AP-1, and TGF-β/Smad Signaling Pathways in UVB-Aged Normal Human Dermal Fibroblasts

    doi: 10.1089/rej.2017.1971

    Figure Lengend Snippet: Inhibitory effect of PVE on MAPKs (A–D) and AP-1 (E–G) . NHDFs were irradiated with UVB (144 mJ/cm 2 ) followed by treatment with PVE (1, 10, 100 μg/mL) for 1.5 hours. Phosphorylation and nonphosphorylation of JNK, ERK, p38, c-Fos, and c-Jun were measured. The band intensities were quantified by densitometry, normalized to the level of β-actin, and calculated as the percentage of the basal response. Values are mean ± SD. ## p

    Article Snippet: Antibodies against ERK, phosphor-ERK, JNK, phosphor-JNK, p38, and phosphor-p38, as well as anti-rabbit-HRP and anti-mouse-HRP antibodies were purchased from Cell Signaling Technology, and those against NF-κB p65, c-Fos, phosphor-c-Fos, c-Jun, phosphor-c-Jun, TGF-β1, Smad2/3, phosphor-Smad2/3, Smad7, and β-actin were purchased from Santa Cruz Biotechnology (Dallas).

    Techniques: Irradiation

    Effect of NF-κB-, p38-, JNK- and ERK-specific inhibitors on OmpA-stimulated production of IL-6 and IL-10. ( A-D ) B cells were purified from spleen, cultured for 1 h with vehicle or the inhibitors of NF-κB (SN50; 100 µg/ml), A , p38 (SB203580; 5 µM), B , JNK (SP600125; 5 µM), C , MEK1/2 (U0126; 5 µM) D , followed by incubation with OmpA (5 µg/ml). After 72 h of culture, the IL-6 and IL-10 levels in the cell supernatants were determined via sandwich ELISA. The data are the mean ± S.E.M of three independent experiments. **, p

    Journal: PLoS ONE

    Article Title: Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase, ERK and NF-κB

    doi: 10.1371/journal.pone.0109107

    Figure Lengend Snippet: Effect of NF-κB-, p38-, JNK- and ERK-specific inhibitors on OmpA-stimulated production of IL-6 and IL-10. ( A-D ) B cells were purified from spleen, cultured for 1 h with vehicle or the inhibitors of NF-κB (SN50; 100 µg/ml), A , p38 (SB203580; 5 µM), B , JNK (SP600125; 5 µM), C , MEK1/2 (U0126; 5 µM) D , followed by incubation with OmpA (5 µg/ml). After 72 h of culture, the IL-6 and IL-10 levels in the cell supernatants were determined via sandwich ELISA. The data are the mean ± S.E.M of three independent experiments. **, p

    Article Snippet: For inhibition studies, B cells were incubated either with TLR2 blocking antibody (purified monoclonal anti-mouse TLR2 antibody [Clone: T2.5]; InvivoGen), Mouse IgG1 isotype control, TLR4 blocking antibody (purified monoclonal antibody to mouse TLR4/MD2 [Clone: T2.5]; InvivoGen), Rat IgG2a isotype control, NF-κB nuclear translocation inhibitor (SN50 peptide; Santa Cruz Biotechnology Inc.), PTK inhibitor (Herbimycin A; Santa Cruz Biotechnology Inc.), p38 inhibitor (SB203580; Cell Signaling Technology), JNK inhibitor (SP600125; Cell Signaling Technology) or MEK1/2 inhibitor (U0126; Cell Signaling Technology) for 1 h prior to incubation with OmpA.

    Techniques: Purification, Cell Culture, Incubation, Sandwich ELISA

    Activation of ERK is critical for OmpA-induced differentiation of B cells into ASCs. B cells were purified from spleen, cultured for 1 h with vehicle or the inhibitors of MEK1/2 (U0126; 5 µM) A , NF-κB (SN50; 100 µg/ml), B , p38 (SB203580; 5 µM), C , JNK (SP600125; 5 µM), D , followed by incubation with OmpA (5 µg/ml) for 72 h. The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT. Number of ASCs/10 5 B cells. The results are the mean ± S.E.M of three independent experiments. ***, p

    Journal: PLoS ONE

    Article Title: Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase, ERK and NF-κB

    doi: 10.1371/journal.pone.0109107

    Figure Lengend Snippet: Activation of ERK is critical for OmpA-induced differentiation of B cells into ASCs. B cells were purified from spleen, cultured for 1 h with vehicle or the inhibitors of MEK1/2 (U0126; 5 µM) A , NF-κB (SN50; 100 µg/ml), B , p38 (SB203580; 5 µM), C , JNK (SP600125; 5 µM), D , followed by incubation with OmpA (5 µg/ml) for 72 h. The total number of ASCs (IgM + IgG-secreting cells) was analyzed by ELISPOT. Number of ASCs/10 5 B cells. The results are the mean ± S.E.M of three independent experiments. ***, p

    Article Snippet: For inhibition studies, B cells were incubated either with TLR2 blocking antibody (purified monoclonal anti-mouse TLR2 antibody [Clone: T2.5]; InvivoGen), Mouse IgG1 isotype control, TLR4 blocking antibody (purified monoclonal antibody to mouse TLR4/MD2 [Clone: T2.5]; InvivoGen), Rat IgG2a isotype control, NF-κB nuclear translocation inhibitor (SN50 peptide; Santa Cruz Biotechnology Inc.), PTK inhibitor (Herbimycin A; Santa Cruz Biotechnology Inc.), p38 inhibitor (SB203580; Cell Signaling Technology), JNK inhibitor (SP600125; Cell Signaling Technology) or MEK1/2 inhibitor (U0126; Cell Signaling Technology) for 1 h prior to incubation with OmpA.

    Techniques: Activation Assay, Purification, Cell Culture, Incubation, Enzyme-linked Immunospot

    OmpA stimulates phosphorylation of protein tyrosine kinases, ERK, JNK and p38 and induces NF-κB activation. B cells were incubated with 5 µg/ml of OmpA for the indicated times, and cell lysates were probed for phosphotyrosine (pY) ( A ), and phosphorylated and total ERK, JNK and p38 ( B ). Representative blots from three independent experiments are shown. Arrows in the pY blot show bands with higher tyrosine phosphorylation. ( C ) B cells were purified from the spleen of BALB'/c mice, stimulated with OmpA (5 µg/ml) for the indicated times, and lysates were probed with phosphorylated and total IκBα. β-Actin was used as an internal control. The data is representative of three independent experiments. ( D ) B cells were cultured in absence and presence of OmpA for 30 min. The cytoplasmic (Cyto) and nuclear (Nuc) extracts were analyzed for p65, Lamin B1 (nuclear marker) and α-tubulin (Cytoplasmic marker) by Western blot analysis. The data shown are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces TLR2-Mediated Activation of B Cells: Involvement of Protein Tyrosine Kinase, ERK and NF-κB

    doi: 10.1371/journal.pone.0109107

    Figure Lengend Snippet: OmpA stimulates phosphorylation of protein tyrosine kinases, ERK, JNK and p38 and induces NF-κB activation. B cells were incubated with 5 µg/ml of OmpA for the indicated times, and cell lysates were probed for phosphotyrosine (pY) ( A ), and phosphorylated and total ERK, JNK and p38 ( B ). Representative blots from three independent experiments are shown. Arrows in the pY blot show bands with higher tyrosine phosphorylation. ( C ) B cells were purified from the spleen of BALB'/c mice, stimulated with OmpA (5 µg/ml) for the indicated times, and lysates were probed with phosphorylated and total IκBα. β-Actin was used as an internal control. The data is representative of three independent experiments. ( D ) B cells were cultured in absence and presence of OmpA for 30 min. The cytoplasmic (Cyto) and nuclear (Nuc) extracts were analyzed for p65, Lamin B1 (nuclear marker) and α-tubulin (Cytoplasmic marker) by Western blot analysis. The data shown are representative of three independent experiments.

    Article Snippet: For inhibition studies, B cells were incubated either with TLR2 blocking antibody (purified monoclonal anti-mouse TLR2 antibody [Clone: T2.5]; InvivoGen), Mouse IgG1 isotype control, TLR4 blocking antibody (purified monoclonal antibody to mouse TLR4/MD2 [Clone: T2.5]; InvivoGen), Rat IgG2a isotype control, NF-κB nuclear translocation inhibitor (SN50 peptide; Santa Cruz Biotechnology Inc.), PTK inhibitor (Herbimycin A; Santa Cruz Biotechnology Inc.), p38 inhibitor (SB203580; Cell Signaling Technology), JNK inhibitor (SP600125; Cell Signaling Technology) or MEK1/2 inhibitor (U0126; Cell Signaling Technology) for 1 h prior to incubation with OmpA.

    Techniques: Activation Assay, Incubation, Purification, Mouse Assay, Cell Culture, Marker, Western Blot

    Depletion of TNFAIP8 affects cellular apoptosis and the p38 mitogen-activated protein kinase signaling pathway. (A) Caspase-3 and caspase-8 activation in control HeLa cells or in TNFAIP8-silenced cells, in the presence or absence of cisplatin (5 µg/ml). (B) Expression levels of p38, p-p38, Bcl-2 and TNFAIP8 in TNFAIP8-silenced HeLa and control cells in the presence or absence of CDDP (5 µg/ml). Data are representative of three independent experiments. TNFAIP8, tumor necrosis factor α-induced protein 8; Scr, scrambled control RNA; CDDP, cisplatin; Bcl-2, B-cell lymphoma 2; p-, phosphorylated.

    Journal: Oncology Letters

    Article Title: TNFAIP8 promotes cisplatin resistance in cervical carcinoma cells by inhibiting cellular apoptosis

    doi: 10.3892/ol.2019.10076

    Figure Lengend Snippet: Depletion of TNFAIP8 affects cellular apoptosis and the p38 mitogen-activated protein kinase signaling pathway. (A) Caspase-3 and caspase-8 activation in control HeLa cells or in TNFAIP8-silenced cells, in the presence or absence of cisplatin (5 µg/ml). (B) Expression levels of p38, p-p38, Bcl-2 and TNFAIP8 in TNFAIP8-silenced HeLa and control cells in the presence or absence of CDDP (5 µg/ml). Data are representative of three independent experiments. TNFAIP8, tumor necrosis factor α-induced protein 8; Scr, scrambled control RNA; CDDP, cisplatin; Bcl-2, B-cell lymphoma 2; p-, phosphorylated.

    Article Snippet: Subsequent to blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with the following primary antibodies at 4°C overnight: TNFAIP8 (1:1,000; ab195810; Abcam); β-actin (1:20,000; a1978; Sigma-Aldrich; Merck KGaA); procaspase-3 and cleaved caspase3 (9662), cleaved caspase-8 (9496), phosphorylated-p38 (9211), p-38 (9212) and Bcl-2 (4223) at 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Activation Assay, Expressing

    p38MAP kinase mediates the NET4/Tmem53 effects on cell cycle proteins. MRC5 and U2OS cells transfected with either siRNA oligos or controls were treated 24 h after transfection with a specific inhibitor of the p38MAP kinase. (A) Protein lysates were generated after a further 48 h and analyzed by Western blot for levels of p53, phosphorylated p38, and p21. This experiment was repeated 3 times and a representative Western blot is shown. (B) Levels of p53, p21 and phosphorylated p38 were quantified from three separate experiments analyzed by LI-COR using fluorescent secondary antibodies and are plotted normalized to the actin control with standard errors. In all cases the p38MAP kinase inhibitor blocked the effects of NET4/TMEM53 knockdown on levels of these cell cycle proteins. (C) The change in the percentage of Ki-67 positive cells induced by NET4/TMEM53 knockdown is partially abrogated by blocking the function of p38 kinase. (D) Effects of NET4/TMEM53 knockdown on senescence, as determined by ß-galactosidase staining, are abrogated by blocking p38MAP kinase function.

    Journal: PLoS ONE

    Article Title: A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal

    doi: 10.1371/journal.pone.0018762

    Figure Lengend Snippet: p38MAP kinase mediates the NET4/Tmem53 effects on cell cycle proteins. MRC5 and U2OS cells transfected with either siRNA oligos or controls were treated 24 h after transfection with a specific inhibitor of the p38MAP kinase. (A) Protein lysates were generated after a further 48 h and analyzed by Western blot for levels of p53, phosphorylated p38, and p21. This experiment was repeated 3 times and a representative Western blot is shown. (B) Levels of p53, p21 and phosphorylated p38 were quantified from three separate experiments analyzed by LI-COR using fluorescent secondary antibodies and are plotted normalized to the actin control with standard errors. In all cases the p38MAP kinase inhibitor blocked the effects of NET4/TMEM53 knockdown on levels of these cell cycle proteins. (C) The change in the percentage of Ki-67 positive cells induced by NET4/TMEM53 knockdown is partially abrogated by blocking the function of p38 kinase. (D) Effects of NET4/TMEM53 knockdown on senescence, as determined by ß-galactosidase staining, are abrogated by blocking p38MAP kinase function.

    Article Snippet: Antibodies Antibodies to the following proteins were used: Ki-67 (610968, BD Transduction Lab), total Rb (4H1 9309, Cell signaling), phospho-Rb (9307, Cell Signaling), p21 (556430, BD Transduction lab), p53 rabbit (9282, Cell signaling), p53 mouse (NCL-p53-DO1, Leica), p38 total (9212, Cell Signaling), active p38 (V3281 Anti-active MAPK Family Sampler, Promega), cyclin E mAb clone HE12 (32-1600, Invitrogen), cyclin A mAb clone Cy-A1 (4710, Sigma), cyclin D (2922, Cell Signaling), cyclin B (SC245, Santa Cruz), LAP2ß (06-1002, Millipore), Lap2α previously described in , Lamin A and B1 (3262 and 3931) previously described in .

    Techniques: Transfection, Generated, Western Blot, Blocking Assay, Staining

    The p-p38, p-JNK, and p-ERK levels in crushed sciatic nerves from WT and αBC −/− mice. Western blot levels of p-p38, p-JNK, and p-ERK in nerves from naïve (N) and 1-, 3-, 5-, and 7-d postcrushed WT and αBC −/− animals (one experiment; n = 2–4 per group). All data represent mean ± SEM. P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: AlphaB-crystallin regulates remyelination after peripheral nerve injury

    doi: 10.1073/pnas.1612136114

    Figure Lengend Snippet: The p-p38, p-JNK, and p-ERK levels in crushed sciatic nerves from WT and αBC −/− mice. Western blot levels of p-p38, p-JNK, and p-ERK in nerves from naïve (N) and 1-, 3-, 5-, and 7-d postcrushed WT and αBC −/− animals (one experiment; n = 2–4 per group). All data represent mean ± SEM. P

    Article Snippet: Membranes were immunoblotted overnight at 4 °C with the following primary antibodies: Ms anti–GAP-43 (MAB347; 1:400; Millipore), Rb anti-αBC (ABN185; 1:1,000; EMD Millipore), Rb anti-actin (A2006; 1:1,000; Sigma-Aldrich), Rb neuregulin 1 Type I (sc-348; 1:500; Santa Cruz), Ms neuregulin 1 Type III (MABN42; 1:1,000; Millipore), Sh ErbB2 (AF5176; 1:1,000; R & D), Ms p-ErbB2 (04–294; 1:1,000; Millipore), Rb AKT (9272; 1:1,000; Cell Signaling), Rb p-AKT (4060; 1:1,000; Cell Signaling), Rb p38 (9212; 1:1,000; Cell Signaling), Ms p-p38 (9216; 1:2,000; Cell Signaling), Rb ERK (9102; 1:1,000; Cell Signaling ), Rb p-ERK (9101; 1:1,000; Cell Signaling ), Rb JNK (9252; 1:1,000; Cell Signaling ), and Rb p-JNK (9251; 1:1,000; Cell Signaling).

    Techniques: Mouse Assay, Western Blot

    NGR1 inhibits serum-induced hCASMC proliferation and migration specifically through PI3K/Akt signaling pathway. ( A ) Western blot analysis of p-Akt/Akt, p-ERK/ERK, p-p38/p38, p-JNK/JNK in hCASMCs treated with NGR1 for 24 h and stimulated with 10% FBS for 0–30 min. Hsp90 was used as loading control. ( B ) Densitometry analysis of the phosphorylation protein normalized to total protein levels. Quiescent hCASMCs were treated with NGR1 (10 μM) for 24 h and incubated with LY294002 (20 μM) or LY294002 and NGR1 in presence or absence serum for 48 h. ( C ) Cell proliferation was quantified as fold increase of control. ( D ) Cell migration was quantified as percentage of area of migrated cells covered area relative to time 0. ( E ) Western blot of pAkt and total Akt from hCASMC treated with FBS, FBS + NGR1 with or without LY294002. ( F ) Densitometry analysis of the phosphorylation protein normalized to total protein levels of Akt under indicated condition. Data shown are means ± SEM. * P

    Journal: Scientific Reports

    Article Title: Notoginsenoside R1 inhibits vascular smooth muscle cell proliferation, migration and neointimal hyperplasia through PI3K/Akt signaling

    doi: 10.1038/s41598-018-25874-y

    Figure Lengend Snippet: NGR1 inhibits serum-induced hCASMC proliferation and migration specifically through PI3K/Akt signaling pathway. ( A ) Western blot analysis of p-Akt/Akt, p-ERK/ERK, p-p38/p38, p-JNK/JNK in hCASMCs treated with NGR1 for 24 h and stimulated with 10% FBS for 0–30 min. Hsp90 was used as loading control. ( B ) Densitometry analysis of the phosphorylation protein normalized to total protein levels. Quiescent hCASMCs were treated with NGR1 (10 μM) for 24 h and incubated with LY294002 (20 μM) or LY294002 and NGR1 in presence or absence serum for 48 h. ( C ) Cell proliferation was quantified as fold increase of control. ( D ) Cell migration was quantified as percentage of area of migrated cells covered area relative to time 0. ( E ) Western blot of pAkt and total Akt from hCASMC treated with FBS, FBS + NGR1 with or without LY294002. ( F ) Densitometry analysis of the phosphorylation protein normalized to total protein levels of Akt under indicated condition. Data shown are means ± SEM. * P

    Article Snippet: Primary antibodies against Akt/phospho-Akt, ERK/phospho-ERK, p38/ phospho-p38 and JNK/ phospho-JNK were all purchased from Cell Signaling Technology (Massachusetts, USA).

    Techniques: Migration, Western Blot, Incubation

    Effects of Hc-CATH on LPS-induced inflammatory response pathways. Western blot of phosphorylation of ERK, JNK, and p38 and the translocation of the NF-κB p65 subunit from cytoplasm to nucleus in MPM cells. MPM cells were co-incubated with LPS

    Journal: The Journal of Biological Chemistry

    Article Title: Identification and Characterization of the First Cathelicidin from Sea Snakes with Potent Antimicrobial and Anti-inflammatory Activity and Special Mechanism *

    doi: 10.1074/jbc.M115.642645

    Figure Lengend Snippet: Effects of Hc-CATH on LPS-induced inflammatory response pathways. Western blot of phosphorylation of ERK, JNK, and p38 and the translocation of the NF-κB p65 subunit from cytoplasm to nucleus in MPM cells. MPM cells were co-incubated with LPS

    Article Snippet: Primary antibodies of phospho-ERK/ERK, phospho-JNK/JNK, phospho-p38/p38, NF-κB p65 (1:2000, Cell Signaling Technology), and GAPDH (1:2000, Dingguo Biotech) were used in Western blot analysis.

    Techniques: Western Blot, Translocation Assay, Incubation

    p38α mediates phosphorylation of KMT1A during myoblasts differentiation. a Western blot analysis of cell extracts from C2-4RE-luc/KMT1A-F expressing control vector (Em), MKK6EE or MKK6DN via lentiviral delivery grown in GM, and C2C12 grown in DM as positive control of differentiation, probed with antibodies for indicated proteins, and β-actin as for loading control. Phosphorylated KMT1A was detected C2-4RE-luc cells expressing only MKK6EE in GM and C2C12 cells in DM. b Western blot analysis of cell extracts from C2C12 grown in GM or DM at various time points, probed with antibodies for detecting indicated proteins, GAPDH as loading control. An increased level of phosphorylated KMT1A was observed with increased activated p38 status during the differentiation of C2C12 myoblasts. c Western blot analysis of cell extracts from C2C12 grown in GM or DM in the presence or absence of SB, probed with antibodies against indicated proteins. GAPDH served as loading control. Inhibition of KMT1A phosphorylation was observed by SB-treatment-induced blockade of p38a activation in cells under DM conditions. d Western blot analysis of cell extracts from C2C12 cells expressing scramble control shRNA (Ctrl) or p38αshRNA via lentiviral delivery grown as specified in GM or DM, probed with antibodies for indicated proteins, where GAPDH for loading control. Inhibition of KMT1A phosphorylation was observed by p38α knockdown. e Control IgG or anti-Flag (M2) immunoprecipitates were retrieved as indicated from extracts of 293A cells expressing with or without Flag-KMT1A via lentiviral delivery. These immunoprecipitates were then subjected to in vitro kinase assay in the presence of purified p38α with or without SB. Subsequently, the reaction mixture was divided equally and evaluated one part for autoradiography and another part for western blot analysis. Phosphorylation of KMT1A directly by p38α was observed

    Journal: Skeletal Muscle

    Article Title: p38α MAPK disables KMT1A-mediated repression of myogenic differentiation program

    doi: 10.1186/s13395-016-0100-z

    Figure Lengend Snippet: p38α mediates phosphorylation of KMT1A during myoblasts differentiation. a Western blot analysis of cell extracts from C2-4RE-luc/KMT1A-F expressing control vector (Em), MKK6EE or MKK6DN via lentiviral delivery grown in GM, and C2C12 grown in DM as positive control of differentiation, probed with antibodies for indicated proteins, and β-actin as for loading control. Phosphorylated KMT1A was detected C2-4RE-luc cells expressing only MKK6EE in GM and C2C12 cells in DM. b Western blot analysis of cell extracts from C2C12 grown in GM or DM at various time points, probed with antibodies for detecting indicated proteins, GAPDH as loading control. An increased level of phosphorylated KMT1A was observed with increased activated p38 status during the differentiation of C2C12 myoblasts. c Western blot analysis of cell extracts from C2C12 grown in GM or DM in the presence or absence of SB, probed with antibodies against indicated proteins. GAPDH served as loading control. Inhibition of KMT1A phosphorylation was observed by SB-treatment-induced blockade of p38a activation in cells under DM conditions. d Western blot analysis of cell extracts from C2C12 cells expressing scramble control shRNA (Ctrl) or p38αshRNA via lentiviral delivery grown as specified in GM or DM, probed with antibodies for indicated proteins, where GAPDH for loading control. Inhibition of KMT1A phosphorylation was observed by p38α knockdown. e Control IgG or anti-Flag (M2) immunoprecipitates were retrieved as indicated from extracts of 293A cells expressing with or without Flag-KMT1A via lentiviral delivery. These immunoprecipitates were then subjected to in vitro kinase assay in the presence of purified p38α with or without SB. Subsequently, the reaction mixture was divided equally and evaluated one part for autoradiography and another part for western blot analysis. Phosphorylation of KMT1A directly by p38α was observed

    Article Snippet: Antibodies used were phospho-p38 (Cell Signaling 9215), β-actin-peroxidase (Sigma A3854), Flag-M2 (Sigma F3165), myogenin (BD Pharmingen 556358), KMT1A (Cell Signaling 8729, and Millipore 07-550 and 05-615), MyoD (Santa Cruz sc-760 and BD Pharmingen 554130), p38α (Cell Signaling 9790), HA-peroxidase (Sigma H6533), acetyl-histone H3 (Millipore, 06-599), trimethyl-histone H3 (Lys-9) (Millipore 07-442), trimethyl-histone H3 (Lys27) (Millipore 07-449), GAPDH (Biodesign H86504M), Brg-1 (Santa Cruz sc-10768), p21cip1 (Santa Cruz sc-397), myosin heavy chain (Developmental Studies Hybridoma Bank, MF-20), total p38 (Cell Signaling 9212), and normal rabbit IgG (Santa Cruz sc-2027).

    Techniques: Western Blot, Expressing, Plasmid Preparation, Positive Control, Inhibition, Activation Assay, shRNA, In Vitro, Kinase Assay, Purification, Autoradiography

    GALR2 stimulates cytokine secretion and angiogenesis via RAP1B-p38-MAPK

    Journal: Molecular cancer therapeutics

    Article Title: The G-protein Coupled Receptor GALR2 Promotes Angiogenesis in Head and Neck Cancer

    doi: 10.1158/1535-7163.MCT-13-0904

    Figure Lengend Snippet: GALR2 stimulates cytokine secretion and angiogenesis via RAP1B-p38-MAPK

    Article Snippet: Cell lysates were electrophoresed ( ) and incubated with primary antibodies (concentration 1:1000 unless otherwise indicated): phospho-p38 (p-p38), total p38 (p38), actin, RAP1B (Cell Signaling), TTP (SantaCruz), p-Serine (Abcam), IL-6 (R & D Systems), GALR2 (Alpha Diagnostics) and GAPDH (Millipore/Upstate).

    Techniques:

    GALR2 induces p38-mediated inhibition of TTP to enhance angiogenesis

    Journal: Molecular cancer therapeutics

    Article Title: The G-protein Coupled Receptor GALR2 Promotes Angiogenesis in Head and Neck Cancer

    doi: 10.1158/1535-7163.MCT-13-0904

    Figure Lengend Snippet: GALR2 induces p38-mediated inhibition of TTP to enhance angiogenesis

    Article Snippet: Cell lysates were electrophoresed ( ) and incubated with primary antibodies (concentration 1:1000 unless otherwise indicated): phospho-p38 (p-p38), total p38 (p38), actin, RAP1B (Cell Signaling), TTP (SantaCruz), p-Serine (Abcam), IL-6 (R & D Systems), GALR2 (Alpha Diagnostics) and GAPDH (Millipore/Upstate).

    Techniques: Inhibition

    Determination of signaling pathways after Nrf2 knockdown in mouse heart. (a, b) Myr treatment inhibited the phosphorylation and nuclei translocation of p65; (c, d) Myr treatment inhibited the phosphorylation of TAK1/p38/JNK1/2 after Nrf2 knockdown, n = 6, ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Myricetin Alleviates Pathological Cardiac Hypertrophy via TRAF6/TAK1/MAPK and Nrf2 Signaling Pathway

    doi: 10.1155/2019/6304058

    Figure Lengend Snippet: Determination of signaling pathways after Nrf2 knockdown in mouse heart. (a, b) Myr treatment inhibited the phosphorylation and nuclei translocation of p65; (c, d) Myr treatment inhibited the phosphorylation of TAK1/p38/JNK1/2 after Nrf2 knockdown, n = 6, ∗ p

    Article Snippet: The following primary antibodies were used in this study: T-ERK (CST, 4695), p-JNK (T183/Y185) (CST, 4668P), T-JNK (CST, 9258S), T-TAK1 (CST, 4060), p-P38 (CST, 4511P), T-p38 (CST, 9212P), p-TAK1 (CST, 4508), p-P65 (s276) (BIOWORLD, BS4135), T-P65 (CST, 8242), GAPDH (CST, 2118), p-ERK1/2 (Thr202/Tyr 204) (CST, 4370P), Histone-3 (Abcam, ab5176), TRAF6 (Santa Cruz, sc-8409), ubiquitin (Proteintech, 10201-2-AP), Nrf2 (Proteintech, 16396-1-AP), 4-hydroxynonenal (Abcam, ab46545), SOD1 (Abcam, ab16831), and catalase (Proteintech, 19792-1-AP).

    Techniques: Translocation Assay

    Protein levels of signaling pathways after Myr treatment. (a, b) Myr inhibited the Nrf2/HO-1 pathway; (c, d) Myr inhibited the phosphorylation and nuclei translocation of p65; (d, e) Myr inhibited the phosphorylation of p38 and JNK1.2 MAPK kinase. n = 6, ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Myricetin Alleviates Pathological Cardiac Hypertrophy via TRAF6/TAK1/MAPK and Nrf2 Signaling Pathway

    doi: 10.1155/2019/6304058

    Figure Lengend Snippet: Protein levels of signaling pathways after Myr treatment. (a, b) Myr inhibited the Nrf2/HO-1 pathway; (c, d) Myr inhibited the phosphorylation and nuclei translocation of p65; (d, e) Myr inhibited the phosphorylation of p38 and JNK1.2 MAPK kinase. n = 6, ∗ p

    Article Snippet: The following primary antibodies were used in this study: T-ERK (CST, 4695), p-JNK (T183/Y185) (CST, 4668P), T-JNK (CST, 9258S), T-TAK1 (CST, 4060), p-P38 (CST, 4511P), T-p38 (CST, 9212P), p-TAK1 (CST, 4508), p-P65 (s276) (BIOWORLD, BS4135), T-P65 (CST, 8242), GAPDH (CST, 2118), p-ERK1/2 (Thr202/Tyr 204) (CST, 4370P), Histone-3 (Abcam, ab5176), TRAF6 (Santa Cruz, sc-8409), ubiquitin (Proteintech, 10201-2-AP), Nrf2 (Proteintech, 16396-1-AP), 4-hydroxynonenal (Abcam, ab46545), SOD1 (Abcam, ab16831), and catalase (Proteintech, 19792-1-AP).

    Techniques: Translocation Assay

    Myr treatment prevented Traf6 ubiquitination in vitro . (a) Myr (20 μ M, preincubation with NRCM for 1 h) treatment prevented PE (50 μ M, 30 min) induced TRAF6 ubiquitination and the phosphorylation of p38 and JNK1/2; (b) Myr treatment inhibited K63-Ub-induced TRAF6 ubiquitination in HEK293 cell; (c) Myr inhibited the interaction between Traf6 and TAK1, and TAK1phosphorylation. All experiments were repeated three times independently.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Myricetin Alleviates Pathological Cardiac Hypertrophy via TRAF6/TAK1/MAPK and Nrf2 Signaling Pathway

    doi: 10.1155/2019/6304058

    Figure Lengend Snippet: Myr treatment prevented Traf6 ubiquitination in vitro . (a) Myr (20 μ M, preincubation with NRCM for 1 h) treatment prevented PE (50 μ M, 30 min) induced TRAF6 ubiquitination and the phosphorylation of p38 and JNK1/2; (b) Myr treatment inhibited K63-Ub-induced TRAF6 ubiquitination in HEK293 cell; (c) Myr inhibited the interaction between Traf6 and TAK1, and TAK1phosphorylation. All experiments were repeated three times independently.

    Article Snippet: The following primary antibodies were used in this study: T-ERK (CST, 4695), p-JNK (T183/Y185) (CST, 4668P), T-JNK (CST, 9258S), T-TAK1 (CST, 4060), p-P38 (CST, 4511P), T-p38 (CST, 9212P), p-TAK1 (CST, 4508), p-P65 (s276) (BIOWORLD, BS4135), T-P65 (CST, 8242), GAPDH (CST, 2118), p-ERK1/2 (Thr202/Tyr 204) (CST, 4370P), Histone-3 (Abcam, ab5176), TRAF6 (Santa Cruz, sc-8409), ubiquitin (Proteintech, 10201-2-AP), Nrf2 (Proteintech, 16396-1-AP), 4-hydroxynonenal (Abcam, ab46545), SOD1 (Abcam, ab16831), and catalase (Proteintech, 19792-1-AP).

    Techniques: In Vitro

    The activation of MAPKs contributes to LQ-mediated apoptotic cell death. (a) 400 μ M LQ treatment resulted in a reduction of the expression of P-ERKs and an increase of the activation of JNK and P38 from 0.5 h to 24 h treatment. (b) The migration of P-ERKs from cytoplasm to nucleus was suppressed by LQ after 3 h exposure. Quantification data of the expressions of P-ERKs, P-JNK, and P-P38 were normalized by corresponding T-ERKs, T-JNK, and T-P38. Data are expressed as mean ± SD ( n = 3) and analyzed using one-way ANOVA. * P

    Journal: BioMed Research International

    Article Title: Liquiritigenin Induces Tumor Cell Death through Mitogen-Activated Protein Kinase- (MPAKs-) Mediated Pathway in Hepatocellular Carcinoma Cells

    doi: 10.1155/2014/965316

    Figure Lengend Snippet: The activation of MAPKs contributes to LQ-mediated apoptotic cell death. (a) 400 μ M LQ treatment resulted in a reduction of the expression of P-ERKs and an increase of the activation of JNK and P38 from 0.5 h to 24 h treatment. (b) The migration of P-ERKs from cytoplasm to nucleus was suppressed by LQ after 3 h exposure. Quantification data of the expressions of P-ERKs, P-JNK, and P-P38 were normalized by corresponding T-ERKs, T-JNK, and T-P38. Data are expressed as mean ± SD ( n = 3) and analyzed using one-way ANOVA. * P

    Article Snippet: The transferred membranes were then blotted with the following primary antibodies at 4°C overnight at dilution of 1 : 1000: phosphor-c-Jun N-terminal kinases (P-JNK), total-JNK (T-JNK), phosphor-P38 (P-P38), total-P38 (T-P38), phosphor-ERKs (P-ERKs), total-ERKs (T-ERKs), cleaved poly (ADP-ribose) polymerase (PARP), Bcl-2, and Bcl-xL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA), followed by treatment with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA).

    Techniques: Activation Assay, Expressing, Migration

    DMF activates phosphorylation of p38 MAP kinase and p38 MAPK inhibition reduces DMF induced HO-1 in ASMC. (A) a representative immuno-blot of the PDGF-BB induced ERK1/2 MAPK (p-ERK1/2) phosphorylation kinetic in the presence and absence of DMF. Similar results were obtained in three additional cell lines. (B) a representative immuno-blot of the kinetic of PDGF-BB induced ERK1/2 MAPK (p-ERK1/2) phosphorylation and its enhancement by DMF; similar results were obtained in three cell lines. (C) a representative immuno-blot of DMF-induced HO-1 expression and its reduction by the p38 MAPK inhibitor SB203580 Similar results were obtained in three cell lines.

    Journal: Respiratory Research

    Article Title: DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1

    doi: 10.1186/1465-9921-11-145

    Figure Lengend Snippet: DMF activates phosphorylation of p38 MAP kinase and p38 MAPK inhibition reduces DMF induced HO-1 in ASMC. (A) a representative immuno-blot of the PDGF-BB induced ERK1/2 MAPK (p-ERK1/2) phosphorylation kinetic in the presence and absence of DMF. Similar results were obtained in three additional cell lines. (B) a representative immuno-blot of the kinetic of PDGF-BB induced ERK1/2 MAPK (p-ERK1/2) phosphorylation and its enhancement by DMF; similar results were obtained in three cell lines. (C) a representative immuno-blot of DMF-induced HO-1 expression and its reduction by the p38 MAPK inhibitor SB203580 Similar results were obtained in three cell lines.

    Article Snippet: Membranes were incubated with blocking buffer (5% w/v non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20) for 1 h at room temperature and were then incubated with one of the following primary antibodies: anti-p38, anti-phospho-p38, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK (all Cell Signalling Technology, Beverly, MA), anti-HO-1 (Calbiochem, Luzern, Switzerland) anti-α-Tubulin (Santa Cruz, Santa Cruz, USA).

    Techniques: Inhibition, Expressing

    GSH reverses the effects of DMF on p38 MAPK phosphorylation, on HO-1 expression and on ASMC proliferation. (A) a representative immuno-blots of the reversing effect of GSH on DMF- and PDGF-BB induced of p38 MAPK phosphorylation in ASMC at 30 min. Similar results were obtained in three cell lines.. (B) a representative immuno-blot of the reversing effect of GSH on the DMF-induced HO-1 expression at 24 h; similar results were obtained in three additional cell lines.. (C) a counteractive effect of GSH on DMF dependent inhibition of ASMC proliferation. Similar results were obtained in four cell lines. Data represents mean ± SEM (unpaired student's t-test).

    Journal: Respiratory Research

    Article Title: DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1

    doi: 10.1186/1465-9921-11-145

    Figure Lengend Snippet: GSH reverses the effects of DMF on p38 MAPK phosphorylation, on HO-1 expression and on ASMC proliferation. (A) a representative immuno-blots of the reversing effect of GSH on DMF- and PDGF-BB induced of p38 MAPK phosphorylation in ASMC at 30 min. Similar results were obtained in three cell lines.. (B) a representative immuno-blot of the reversing effect of GSH on the DMF-induced HO-1 expression at 24 h; similar results were obtained in three additional cell lines.. (C) a counteractive effect of GSH on DMF dependent inhibition of ASMC proliferation. Similar results were obtained in four cell lines. Data represents mean ± SEM (unpaired student's t-test). "V" indicates the drug's vehicle 0.05% DMSO. (D) down regulation of HO-1 by a respective siRNAs counteracted the anti-proliferative effect of DMF. Data represents the mean ± SEM of 9 independent experiments performed in 3 ASMC lines. Statistics have been calculated by Wilcoxon-Mann-Whitney U-test.

    Article Snippet: Membranes were incubated with blocking buffer (5% w/v non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20) for 1 h at room temperature and were then incubated with one of the following primary antibodies: anti-p38, anti-phospho-p38, anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK, anti-phospho-JNK (all Cell Signalling Technology, Beverly, MA), anti-HO-1 (Calbiochem, Luzern, Switzerland) anti-α-Tubulin (Santa Cruz, Santa Cruz, USA).

    Techniques: Expressing, Western Blot, Inhibition, MANN-WHITNEY

    c-N-Ras downregulates p38 activation and activity following the induction of apoptosis. (A) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN43A] were left untreated or treated for varying times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. A 50-μg portion of each lysate was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer, the blot was developed with anti-phospho-p38 (pp38) and anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) N-Ras knockout and control cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to immunoprecipitate p38 as described in Materials and Methods. The resulting immunocomplexes were washed and used in a kinase assay as described in the text with 25 μCi of [γ- 32 P]ATP per reaction and GST-ATF2 as the substrate. Signals were detected with a PhosphorImager. The nonimmune control (NI) consisted of immunoprecipitation of 1 h treated N +/+ (1) control cells using an equal amount of nonimmune rabbit serum. The results are representative of four separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to analyze total levels of p38 using anti-p38 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. The results are representative of two separate experiments. (D) N-Ras knockout cells [N −/− (3) cell line], control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (3)/wtN5] were left untreated in complete medium or starved of serum for various times as described in Materials and Methods. At the indicated times, cell lysates were prepared. A 50-μg portion of each lysates was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer to PVDF, the blot was developed with anti-phospho-p38 and anti-rabbit secondary antibody-HRP as described in the text. Bands were visualized by standard ECL techniques. The results are representative of two separate experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38

    doi:

    Figure Lengend Snippet: c-N-Ras downregulates p38 activation and activity following the induction of apoptosis. (A) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN43A] were left untreated or treated for varying times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. A 50-μg portion of each lysate was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer, the blot was developed with anti-phospho-p38 (pp38) and anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) N-Ras knockout and control cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to immunoprecipitate p38 as described in Materials and Methods. The resulting immunocomplexes were washed and used in a kinase assay as described in the text with 25 μCi of [γ- 32 P]ATP per reaction and GST-ATF2 as the substrate. Signals were detected with a PhosphorImager. The nonimmune control (NI) consisted of immunoprecipitation of 1 h treated N +/+ (1) control cells using an equal amount of nonimmune rabbit serum. The results are representative of four separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to analyze total levels of p38 using anti-p38 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. The results are representative of two separate experiments. (D) N-Ras knockout cells [N −/− (3) cell line], control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (3)/wtN5] were left untreated in complete medium or starved of serum for various times as described in Materials and Methods. At the indicated times, cell lysates were prepared. A 50-μg portion of each lysates was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer to PVDF, the blot was developed with anti-phospho-p38 and anti-rabbit secondary antibody-HRP as described in the text. Bands were visualized by standard ECL techniques. The results are representative of two separate experiments.

    Article Snippet: Phosphospecific Bad polyclonal (Ser136 ), phosphospecific Akt rabbit polyclonal (Ser473 ), phospho-p38 polyclonal, p38 polyclonal (for Western blots), phospho-MKK4 polyclonal, phospho-MKK3/6 polyclonal, and MKK3 polyclonal antibodies were from Cell Signaling Technology (Beverly, Mass.).

    Techniques: Activation Assay, Activity Assay, Knock-Out, Electrophoresis, Kinase Assay, Immunoprecipitation

    c-N-Ras downregulates the upstream kinase activators of JNK and p38. (A) (Upper panel) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN3] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 100 μg of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer to PVDF, the blot was incubated with anti-phospho-MKK4 (pMKK4) polyclonal antibody. The blot was developed by using anti-rabbit secondar y antibody-HRP and standard ECL techniques. The positive control consisted of N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of four separate experiments. (Lower panel) A 100-μg portion of the same lysates as in the upper panel was electrophoresed, transferred, and blotted with anti-MKK4 polyclonal antibody (1:1,000 dilution) followed by anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) (Upper panel) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 100 μg of protein of each lysate was electrophoresed and transferred to PVDF. The blot was developed with anti-phospo-MKK3/6 (pMKK3/6) antibody and anti-rabbit secondary antibody-HRP. Bands were visualized by standard ECL techniques. This experiment is representative of three separate experiments. (Lower panel) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A. Cell lysates (100 μg) were prepared for analysis of total MKK3 levels by using anti-MKK3 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. (C) N-Ras knockout and control N +/+ cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. Cell lysates were prepared at the indicated times, and protein concentrations determined. A 2-μg portion of unactive His 6 -tagged JNK1α1 was incubated with 12 μg of each lysate in the presence of 50 μM unlabeled ATP for 20 min at 30°C. The added His 6 -JNK1α1 was isolated by incubation with nickel resin (ProBond; Invitrogen) for 45 min at 4°C. The resin was washed with p21 buffer and used in a second kinase assay to measure the activity of the isolated, recombinant JNK1α1 by using GST-ATF2 as the substrate as described in Materials and Methods. The unactive JNK1α1 incubated in the absence of lysate had no activity. The results are representative of three separate experiments. (D) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A, and at the indicated times cell lysates were prepared. A 1-μg portion of unactive GST-p38α was incubated for 20 min at 30°C, as described in the text, with 12 μg of each lysate in p21 buffer containing 50 μM ATP. The GST-p38α was isolated with glutathione-agarose, and its activity was measured in a second kinase reaction using [γ- 32 P]ATP with GST-ATF2 as the substrate, as described in Materials and Methods. N-Ras knockout cells were treated for 1 h with 20 μg of anisomycin per ml, and the lysate was used to activate the GST-p38α as a positive control. The unactive GST-p38α had no activity when incubated in the absence of cell lysate. (E) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were passaged into complete medium and 48 h later were either left untreated ( t = 0) or challenged with 10 ng of EGF per ml for the times indicated. Cell lysates prepared at the indicated times were electrophoresed and transferred to PVDF. The membranes were immunoblotted for phospho-JNK (upper panel, 50 μg/lane) and pErk (Santa Cruz Biotechnology; lower panel, 20 μg/lane). The results are representative of three separate experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38

    doi:

    Figure Lengend Snippet: c-N-Ras downregulates the upstream kinase activators of JNK and p38. (A) (Upper panel) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN3] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 100 μg of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer to PVDF, the blot was incubated with anti-phospho-MKK4 (pMKK4) polyclonal antibody. The blot was developed by using anti-rabbit secondar y antibody-HRP and standard ECL techniques. The positive control consisted of N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of four separate experiments. (Lower panel) A 100-μg portion of the same lysates as in the upper panel was electrophoresed, transferred, and blotted with anti-MKK4 polyclonal antibody (1:1,000 dilution) followed by anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) (Upper panel) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 100 μg of protein of each lysate was electrophoresed and transferred to PVDF. The blot was developed with anti-phospo-MKK3/6 (pMKK3/6) antibody and anti-rabbit secondary antibody-HRP. Bands were visualized by standard ECL techniques. This experiment is representative of three separate experiments. (Lower panel) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A. Cell lysates (100 μg) were prepared for analysis of total MKK3 levels by using anti-MKK3 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. (C) N-Ras knockout and control N +/+ cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. Cell lysates were prepared at the indicated times, and protein concentrations determined. A 2-μg portion of unactive His 6 -tagged JNK1α1 was incubated with 12 μg of each lysate in the presence of 50 μM unlabeled ATP for 20 min at 30°C. The added His 6 -JNK1α1 was isolated by incubation with nickel resin (ProBond; Invitrogen) for 45 min at 4°C. The resin was washed with p21 buffer and used in a second kinase assay to measure the activity of the isolated, recombinant JNK1α1 by using GST-ATF2 as the substrate as described in Materials and Methods. The unactive JNK1α1 incubated in the absence of lysate had no activity. The results are representative of three separate experiments. (D) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A, and at the indicated times cell lysates were prepared. A 1-μg portion of unactive GST-p38α was incubated for 20 min at 30°C, as described in the text, with 12 μg of each lysate in p21 buffer containing 50 μM ATP. The GST-p38α was isolated with glutathione-agarose, and its activity was measured in a second kinase reaction using [γ- 32 P]ATP with GST-ATF2 as the substrate, as described in Materials and Methods. N-Ras knockout cells were treated for 1 h with 20 μg of anisomycin per ml, and the lysate was used to activate the GST-p38α as a positive control. The unactive GST-p38α had no activity when incubated in the absence of cell lysate. (E) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were passaged into complete medium and 48 h later were either left untreated ( t = 0) or challenged with 10 ng of EGF per ml for the times indicated. Cell lysates prepared at the indicated times were electrophoresed and transferred to PVDF. The membranes were immunoblotted for phospho-JNK (upper panel, 50 μg/lane) and pErk (Santa Cruz Biotechnology; lower panel, 20 μg/lane). The results are representative of three separate experiments.

    Article Snippet: Phosphospecific Bad polyclonal (Ser136 ), phosphospecific Akt rabbit polyclonal (Ser473 ), phospho-p38 polyclonal, p38 polyclonal (for Western blots), phospho-MKK4 polyclonal, phospho-MKK3/6 polyclonal, and MKK3 polyclonal antibodies were from Cell Signaling Technology (Beverly, Mass.).

    Techniques: Knock-Out, Electrophoresis, Incubation, Positive Control, Isolation, Kinase Assay, Activity Assay, Recombinant

    Inhibition of ASK1 activation by Trx1. ( A ) Representative blot of four experiments from Trx1-ASK1 co-immunoprecipitation. Trx1 binding was decreased in MI heart, which indirectly indicates activation of ASK1. The blot was stripped and re-probed with ASK1 antibody to show equal loading. ( B ) Role of TrxR in IGF-1-mediated recovery of Trx1-ASK1 binding in MI hearts. ASK1-Trx1 interaction was assessed by densitometry. ( C ) Phosphorylation levels of p38 and JNK determined by Western blot in sham hearts perfused with IGF-1. Representative blots are shown to the right. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Role of Apoptosis Signal-Regulating Kinase-1-c-Jun NH2-Terminal Kinase-p38 Signaling in Voltage-Gated K+ Channel Remodeling of the Failing Heart: Regulation by Thioredoxin

    doi: 10.1089/ars.2010.3095

    Figure Lengend Snippet: Inhibition of ASK1 activation by Trx1. ( A ) Representative blot of four experiments from Trx1-ASK1 co-immunoprecipitation. Trx1 binding was decreased in MI heart, which indirectly indicates activation of ASK1. The blot was stripped and re-probed with ASK1 antibody to show equal loading. ( B ) Role of TrxR in IGF-1-mediated recovery of Trx1-ASK1 binding in MI hearts. ASK1-Trx1 interaction was assessed by densitometry. ( C ) Phosphorylation levels of p38 and JNK determined by Western blot in sham hearts perfused with IGF-1. Representative blots are shown to the right. * p

    Article Snippet: Activation of p38 kinase was assessed by a nonradioactive p38 kinase assay kit (Cell Signaling Technology) according to the manufacturer's instructions.

    Techniques: Inhibition, Activation Assay, Immunoprecipitation, Binding Assay, Western Blot

    Upregulation of I to density by JNK and p38 inhibitors. ( A ) Superimposed current traces are shown for myocytes from post-MI hearts untreated or treated with the JNK inhibitor SP600125 (10 (μ M ) for 4–5 h. ( B ) Mean I-V relations of I to illustrate de-remodeling effect of SP600125 or inhibitory peptide JNKI-1 (10 (μ M ). JNKI-Neg, negative control peptide for JNKI-1. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Role of Apoptosis Signal-Regulating Kinase-1-c-Jun NH2-Terminal Kinase-p38 Signaling in Voltage-Gated K+ Channel Remodeling of the Failing Heart: Regulation by Thioredoxin

    doi: 10.1089/ars.2010.3095

    Figure Lengend Snippet: Upregulation of I to density by JNK and p38 inhibitors. ( A ) Superimposed current traces are shown for myocytes from post-MI hearts untreated or treated with the JNK inhibitor SP600125 (10 (μ M ) for 4–5 h. ( B ) Mean I-V relations of I to illustrate de-remodeling effect of SP600125 or inhibitory peptide JNKI-1 (10 (μ M ). JNKI-Neg, negative control peptide for JNKI-1. * p

    Article Snippet: Activation of p38 kinase was assessed by a nonradioactive p38 kinase assay kit (Cell Signaling Technology) according to the manufacturer's instructions.

    Techniques: Negative Control

    JNK and p38 activity post-MI. ( A ) Representative immunoblots from six experiments of sham-operated and MI hearts using anti-p-JNK and anti-p-p38 antibody. The same blot was stripped and re-probed with anti-JNK and anti-p38 antibody to show total JNK and p38. ( B, C ) Comparisons of JNK and p38 kinase activity in post-MI and sham hearts. The in vitro kinase assay measured the phosphorylation of recombinant c-Jun (JNK substrate) or ATF-2 (p38 substrate). The relative kinase activity was determined by densitometry. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Role of Apoptosis Signal-Regulating Kinase-1-c-Jun NH2-Terminal Kinase-p38 Signaling in Voltage-Gated K+ Channel Remodeling of the Failing Heart: Regulation by Thioredoxin

    doi: 10.1089/ars.2010.3095

    Figure Lengend Snippet: JNK and p38 activity post-MI. ( A ) Representative immunoblots from six experiments of sham-operated and MI hearts using anti-p-JNK and anti-p-p38 antibody. The same blot was stripped and re-probed with anti-JNK and anti-p38 antibody to show total JNK and p38. ( B, C ) Comparisons of JNK and p38 kinase activity in post-MI and sham hearts. The in vitro kinase assay measured the phosphorylation of recombinant c-Jun (JNK substrate) or ATF-2 (p38 substrate). The relative kinase activity was determined by densitometry. * p

    Article Snippet: Activation of p38 kinase was assessed by a nonradioactive p38 kinase assay kit (Cell Signaling Technology) according to the manufacturer's instructions.

    Techniques: Activity Assay, Western Blot, In Vitro, Kinase Assay, Recombinant

    Attenuation of JNK and p38 activity by IGF-1. ( A ) Representative Western blot of p-JNK/JNK and p-p38/p38. The same blot was stripped and re-probed with GAPDH to show equal loading. ( B ) JNK activity assay. ( C ) p38 kinase activity assay. The relative kinase activity was determined by densitometry. * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Role of Apoptosis Signal-Regulating Kinase-1-c-Jun NH2-Terminal Kinase-p38 Signaling in Voltage-Gated K+ Channel Remodeling of the Failing Heart: Regulation by Thioredoxin

    doi: 10.1089/ars.2010.3095

    Figure Lengend Snippet: Attenuation of JNK and p38 activity by IGF-1. ( A ) Representative Western blot of p-JNK/JNK and p-p38/p38. The same blot was stripped and re-probed with GAPDH to show equal loading. ( B ) JNK activity assay. ( C ) p38 kinase activity assay. The relative kinase activity was determined by densitometry. * p

    Article Snippet: Activation of p38 kinase was assessed by a nonradioactive p38 kinase assay kit (Cell Signaling Technology) according to the manufacturer's instructions.

    Techniques: Activity Assay, Western Blot, Kinase Assay

    pX-mediated p38 MAPK activation initiates G 2 /M checkpoint in 4pX-1 cells. Flow cytometry analyses of 4pX-1 cells grown for 18 h ± tetracycline addition. Cells were treated with 5 μg of tetracycline/ml (A), not treated with tetracycline (B), or not treated with tetracycline while being treated with 10 μM SB 203580 (C). (D) Western blot analyses of 4pX-1 WCE isolated ± pX for 18 h and ± 10 μM SB 203580. Where indicated, Ser216 phosphorylation of Cdc25C (upper panel) or total Cdc25C (lower panel) was monitored. Quantitation was by the OPTIMAS 6.1 software.

    Journal: Journal of Virology

    Article Title: Hepatitis B Virus X Protein Activates the p38 Mitogen-Activated Protein Kinase Pathway in Dedifferentiated Hepatocytes

    doi: 10.1128/JVI.76.19.9763-9772.2002

    Figure Lengend Snippet: pX-mediated p38 MAPK activation initiates G 2 /M checkpoint in 4pX-1 cells. Flow cytometry analyses of 4pX-1 cells grown for 18 h ± tetracycline addition. Cells were treated with 5 μg of tetracycline/ml (A), not treated with tetracycline (B), or not treated with tetracycline while being treated with 10 μM SB 203580 (C). (D) Western blot analyses of 4pX-1 WCE isolated ± pX for 18 h and ± 10 μM SB 203580. Where indicated, Ser216 phosphorylation of Cdc25C (upper panel) or total Cdc25C (lower panel) was monitored. Quantitation was by the OPTIMAS 6.1 software.

    Article Snippet: For detecting total Stat3 and p38 MAPK, WCE (10 μg) from each time point was analyzed by Western blot analyses employing total Stat3 and p38 antibody (Cell Signaling Technology, Inc.), respectively.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Western Blot, Isolation, Quantitation Assay, Software

    pX activates the p38 MAPK pathway in 4pX-1 cells. (A) Transient-transreporter assays employing pFR-luciferase reporter (100 ng) and pFA-CHOP expression (100 ng) plasmids (Stratagene) in 3pX-1 and 4pX-1 cells ± 5 μg of tetracycline/ml. Inhibitor concentration is as follows: 10 μM SB 203580, 3 μM PP2, and 20 μM BAPTA-AM. (B) In vitro p38 immunocomplex kinase assays with WCE (150 μg) from 4pX-1 and 3pX-1 cells, isolated at the indicated times ± 5 μg of tetracycline/ml. GST-ATF2 (2 μg) substrate phosphorylation was monitored by Western blots using phospho-ATF2 antibodies. Sorbitol treatment was the positive control. Quantitation was performed by digital densitometry using the OPTIMAS 6.1 software. pX-dependent ATF2 phosphorylation was statistically significant ( P

    Journal: Journal of Virology

    Article Title: Hepatitis B Virus X Protein Activates the p38 Mitogen-Activated Protein Kinase Pathway in Dedifferentiated Hepatocytes

    doi: 10.1128/JVI.76.19.9763-9772.2002

    Figure Lengend Snippet: pX activates the p38 MAPK pathway in 4pX-1 cells. (A) Transient-transreporter assays employing pFR-luciferase reporter (100 ng) and pFA-CHOP expression (100 ng) plasmids (Stratagene) in 3pX-1 and 4pX-1 cells ± 5 μg of tetracycline/ml. Inhibitor concentration is as follows: 10 μM SB 203580, 3 μM PP2, and 20 μM BAPTA-AM. (B) In vitro p38 immunocomplex kinase assays with WCE (150 μg) from 4pX-1 and 3pX-1 cells, isolated at the indicated times ± 5 μg of tetracycline/ml. GST-ATF2 (2 μg) substrate phosphorylation was monitored by Western blots using phospho-ATF2 antibodies. Sorbitol treatment was the positive control. Quantitation was performed by digital densitometry using the OPTIMAS 6.1 software. pX-dependent ATF2 phosphorylation was statistically significant ( P

    Article Snippet: For detecting total Stat3 and p38 MAPK, WCE (10 μg) from each time point was analyzed by Western blot analyses employing total Stat3 and p38 antibody (Cell Signaling Technology, Inc.), respectively.

    Techniques: Luciferase, Expressing, Concentration Assay, In Vitro, Isolation, Western Blot, Positive Control, Quantitation Assay, Software

    p38 MAPK phosphorylate HSF1 in vitro . (A) Electrophoretic mobility (NuPAGE NoVex Bis-Tris 10% gel) of recombinant hexahistidine-tagged HSF1 wild-type (WT) and S326A mutant. (B to G) Purified activated recombinant p38 MAPK isoforms (0.06 mU/μl) were incubated with recombinant wild-type (WT), S326A mutant HSF1, MK2, or myelin basic protein (MBP) (all at 0.1 μg/μl) at 30°C for 15 min in the presence of 10 mM MgCl 2 and 0.1 mM [γ- 32 P]ATP. Identical reactions were carried out in the presence of increasing concentrations of the p38α/β inhibitor SB202190 or BIRB0796, which inhibits all p38 isoforms. The reactions were terminated by the addition of SDS gel loading buffer, the samples were loaded on SDS-PAGE, and the excess [γ- 32 P]ATP was removed by electrophoresis. (C and E to G) The gels were dried and subjected to autoradiography. After staining with Coomassie brilliant blue, the protein bands were excised and the incorporated radioactivity (B) was determined by scintillation counting. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

    doi: 10.1128/MCB.00292-16

    Figure Lengend Snippet: p38 MAPK phosphorylate HSF1 in vitro . (A) Electrophoretic mobility (NuPAGE NoVex Bis-Tris 10% gel) of recombinant hexahistidine-tagged HSF1 wild-type (WT) and S326A mutant. (B to G) Purified activated recombinant p38 MAPK isoforms (0.06 mU/μl) were incubated with recombinant wild-type (WT), S326A mutant HSF1, MK2, or myelin basic protein (MBP) (all at 0.1 μg/μl) at 30°C for 15 min in the presence of 10 mM MgCl 2 and 0.1 mM [γ- 32 P]ATP. Identical reactions were carried out in the presence of increasing concentrations of the p38α/β inhibitor SB202190 or BIRB0796, which inhibits all p38 isoforms. The reactions were terminated by the addition of SDS gel loading buffer, the samples were loaded on SDS-PAGE, and the excess [γ- 32 P]ATP was removed by electrophoresis. (C and E to G) The gels were dried and subjected to autoradiography. After staining with Coomassie brilliant blue, the protein bands were excised and the incorporated radioactivity (B) was determined by scintillation counting. *, P

    Article Snippet: Proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and probed with specific antibodies against Hsp70 (mouse monoclonal, 1:1,000; StressMarq, York, United Kingdom), Hsp90 (mouse monoclonal, 1:5,000; BD Biosciences, New Jersey), HER2 (rabbit polyclonal, 1:500; Millipore, CA), RAF1 (rabbit polyclonal, 1:200; Santa Cruz, CA), HSF1 (rabbit polyclonal, 1:1,000; Enzo Life Sciences, Exeter, United Kingdom), pS326-HSF1 (rabbit polyclonal, 1:10,000; Abcam, Cambridge, United Kingdom), p38 MAPK (rabbit polyclonal, 1:1,000; Cell Signaling, MA), pp38 MAPK (rabbit polyclonal, 1:1,000; Cell Signaling), JNK1/2 (rabbit polyclonal, 1:1,000; Cell Signaling), pJNK1/2 (rabbit polyclonal, 1:1,000; Biosource Europe, Nivelles, Belgium), pERK1/2 (rabbit polyclonal, 1:1,000; Cell Signaling), pT334-MK2 (rabbit polyclonal, 1:1,000, Cell Signaling), and pS235/6 S6 (rabbit polyclonal, 1:5,000l Cell Signaling).

    Techniques: In Vitro, Recombinant, Mutagenesis, Purification, Incubation, SDS-Gel, SDS Page, Electrophoresis, Autoradiography, Staining, Radioactivity

    PEITC activates p38 and JNK1/2 MAPK, and inhibits mTOR. MDA-MB-231 cells (2.5 × 10 5 per well) growing in six-well plates were treated with vehicle (0.1% acetonitrile) or PEITC for either 24 h (A and B), 3 h (C and E), or for the indicated periods of time (D). The levels of HSF1, pS326 HSF1, pS235/6 S6, Hsp70, the p38 isoforms α, γ, and δ, phosphorylated p38 (pp38), phosphorylated p38α (pp38α), JNK1/2, and phosphorylated JNK1/2 (JNK1/2) were detected by Western blot analysis.

    Journal: Molecular and Cellular Biology

    Article Title: Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

    doi: 10.1128/MCB.00292-16

    Figure Lengend Snippet: PEITC activates p38 and JNK1/2 MAPK, and inhibits mTOR. MDA-MB-231 cells (2.5 × 10 5 per well) growing in six-well plates were treated with vehicle (0.1% acetonitrile) or PEITC for either 24 h (A and B), 3 h (C and E), or for the indicated periods of time (D). The levels of HSF1, pS326 HSF1, pS235/6 S6, Hsp70, the p38 isoforms α, γ, and δ, phosphorylated p38 (pp38), phosphorylated p38α (pp38α), JNK1/2, and phosphorylated JNK1/2 (JNK1/2) were detected by Western blot analysis.

    Article Snippet: Proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and probed with specific antibodies against Hsp70 (mouse monoclonal, 1:1,000; StressMarq, York, United Kingdom), Hsp90 (mouse monoclonal, 1:5,000; BD Biosciences, New Jersey), HER2 (rabbit polyclonal, 1:500; Millipore, CA), RAF1 (rabbit polyclonal, 1:200; Santa Cruz, CA), HSF1 (rabbit polyclonal, 1:1,000; Enzo Life Sciences, Exeter, United Kingdom), pS326-HSF1 (rabbit polyclonal, 1:10,000; Abcam, Cambridge, United Kingdom), p38 MAPK (rabbit polyclonal, 1:1,000; Cell Signaling, MA), pp38 MAPK (rabbit polyclonal, 1:1,000; Cell Signaling), JNK1/2 (rabbit polyclonal, 1:1,000; Cell Signaling), pJNK1/2 (rabbit polyclonal, 1:1,000; Biosource Europe, Nivelles, Belgium), pERK1/2 (rabbit polyclonal, 1:1,000; Cell Signaling), pT334-MK2 (rabbit polyclonal, 1:1,000, Cell Signaling), and pS235/6 S6 (rabbit polyclonal, 1:5,000l Cell Signaling).

    Techniques: Multiple Displacement Amplification, Western Blot

    Deletion or inhibition of p38γ MAPK reduces the levels of pS326 HSF1 in cells. (A) Immunoblotting for p38 γ and δ in A431 cells, which either express both p38γ and p38δ (WT) or in which p38γ or p38δ had been stably knocked down using selective shRNA. (B) A431 cells (5 × 10 5 per well, WT or p38γ or p38δ deficient) were preincubated for 1 h with vehicle (0.1% acetonitrile) or SB202190, and exposed to heat shock (42°C) for a further 1 h. (C) p38γ or p38δ was stably knocked down in MDA-MB-231 cells using selective shRNA. The levels of total HSF1, HSF1 phosphorylated at S326, total p38, p38γ, and p38δ, and Hsp70 were detected by Western blot analysis. (D and E) MDA-MB-231 cells (5 × 10 5 per well) grown in six-well plates were pretreated with vehicle (0.1% acetonitrile), SB202190, or BIRB0796 for 1 h and subsequently either treated with vehicle (0.1% acetonitrile) or PEITC for a further 1.5 h. HSF1 phosphorylated at S326 (B to E) and MK2 phosphorylated at T334 (D) were detected by Western blot analysis. The levels of α-tubulin (A) or β-actin (B to E) served as loading controls.

    Journal: Molecular and Cellular Biology

    Article Title: Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases

    doi: 10.1128/MCB.00292-16

    Figure Lengend Snippet: Deletion or inhibition of p38γ MAPK reduces the levels of pS326 HSF1 in cells. (A) Immunoblotting for p38 γ and δ in A431 cells, which either express both p38γ and p38δ (WT) or in which p38γ or p38δ had been stably knocked down using selective shRNA. (B) A431 cells (5 × 10 5 per well, WT or p38γ or p38δ deficient) were preincubated for 1 h with vehicle (0.1% acetonitrile) or SB202190, and exposed to heat shock (42°C) for a further 1 h. (C) p38γ or p38δ was stably knocked down in MDA-MB-231 cells using selective shRNA. The levels of total HSF1, HSF1 phosphorylated at S326, total p38, p38γ, and p38δ, and Hsp70 were detected by Western blot analysis. (D and E) MDA-MB-231 cells (5 × 10 5 per well) grown in six-well plates were pretreated with vehicle (0.1% acetonitrile), SB202190, or BIRB0796 for 1 h and subsequently either treated with vehicle (0.1% acetonitrile) or PEITC for a further 1.5 h. HSF1 phosphorylated at S326 (B to E) and MK2 phosphorylated at T334 (D) were detected by Western blot analysis. The levels of α-tubulin (A) or β-actin (B to E) served as loading controls.

    Article Snippet: Proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and probed with specific antibodies against Hsp70 (mouse monoclonal, 1:1,000; StressMarq, York, United Kingdom), Hsp90 (mouse monoclonal, 1:5,000; BD Biosciences, New Jersey), HER2 (rabbit polyclonal, 1:500; Millipore, CA), RAF1 (rabbit polyclonal, 1:200; Santa Cruz, CA), HSF1 (rabbit polyclonal, 1:1,000; Enzo Life Sciences, Exeter, United Kingdom), pS326-HSF1 (rabbit polyclonal, 1:10,000; Abcam, Cambridge, United Kingdom), p38 MAPK (rabbit polyclonal, 1:1,000; Cell Signaling, MA), pp38 MAPK (rabbit polyclonal, 1:1,000; Cell Signaling), JNK1/2 (rabbit polyclonal, 1:1,000; Cell Signaling), pJNK1/2 (rabbit polyclonal, 1:1,000; Biosource Europe, Nivelles, Belgium), pERK1/2 (rabbit polyclonal, 1:1,000; Cell Signaling), pT334-MK2 (rabbit polyclonal, 1:1,000, Cell Signaling), and pS235/6 S6 (rabbit polyclonal, 1:5,000l Cell Signaling).

    Techniques: Inhibition, Stable Transfection, shRNA, Multiple Displacement Amplification, Western Blot

    Regulatory mechanism for differential levels of H3S10ph in GC. a Immunoblot analysis ( upper panel ) of paired tissue ( n = 5) and densitometry-based analysis of relative levels ( lower panel ) showed a significant increase in ph-MSK1 and ph-p38 levels and decrease in ph-ERK1/2 levels in tumor compared to resection margin tissues. b Representative image of immunohistochemistry (×40) ( left panel ) analysis in paired tissue samples ( n = 10) and comparison of their relative H-score ( right panel ) showed high ph-MSK1 levels in tumor than resection margin tissues. c Immunoblot analysis of AGS cells transiently over-expressing MSK1 showed moderate increase of ph-MSK1 and H3S10ph in corresponding lanes. d , e Immunoblot analysis of AGS and KATOII cells and immunofluorescence analysis of AGS cells after 6-h treatment with MSK1 inhibitor, H89 (20 μM) showed loss of ph-MSK1 and H3S10ph. f , g Immunoblot analysis of AGS and KATOII cells and immunofluorescence analysis of AGS cells showed loss of ph-MSK1 and H3S10ph only after 1-h treatment of p38 inhibitor SB203580 (10 μM), but not for ERK1/2 inhibitor PD98059 (10 μM) treatment. GC gastric cancer, RM resection margin either PRM or DRM with maximum distance from the site of the tumor, T tumor, P patient. #Fast green-stained PVDF membrane used in Fig. 4d as well. Statistical tests are done by using Wilcoxon matched pairs test. * p

    Journal: Clinical Epigenetics

    Article Title: p38-MAPK/MSK1-mediated overexpression of histone H3 serine 10 phosphorylation defines distance-dependent prognostic value of negative resection margin in gastric cancer

    doi: 10.1186/s13148-016-0255-9

    Figure Lengend Snippet: Regulatory mechanism for differential levels of H3S10ph in GC. a Immunoblot analysis ( upper panel ) of paired tissue ( n = 5) and densitometry-based analysis of relative levels ( lower panel ) showed a significant increase in ph-MSK1 and ph-p38 levels and decrease in ph-ERK1/2 levels in tumor compared to resection margin tissues. b Representative image of immunohistochemistry (×40) ( left panel ) analysis in paired tissue samples ( n = 10) and comparison of their relative H-score ( right panel ) showed high ph-MSK1 levels in tumor than resection margin tissues. c Immunoblot analysis of AGS cells transiently over-expressing MSK1 showed moderate increase of ph-MSK1 and H3S10ph in corresponding lanes. d , e Immunoblot analysis of AGS and KATOII cells and immunofluorescence analysis of AGS cells after 6-h treatment with MSK1 inhibitor, H89 (20 μM) showed loss of ph-MSK1 and H3S10ph. f , g Immunoblot analysis of AGS and KATOII cells and immunofluorescence analysis of AGS cells showed loss of ph-MSK1 and H3S10ph only after 1-h treatment of p38 inhibitor SB203580 (10 μM), but not for ERK1/2 inhibitor PD98059 (10 μM) treatment. GC gastric cancer, RM resection margin either PRM or DRM with maximum distance from the site of the tumor, T tumor, P patient. #Fast green-stained PVDF membrane used in Fig. 4d as well. Statistical tests are done by using Wilcoxon matched pairs test. * p

    Article Snippet: Proteins on PVDF membrane were hybridized with anti-H3 (Upstate-06-755; 1:2000 dilution), H4 (Millipore-07-108; 1:4000 dilution), H3S10ph (Millipore-06-570; 1:7000 dilution), H4K16ac (Millipore-07-329; 1:8000 dilution), H4K20me3 (Abcam-9053; 1:4000 dilution), β-actin (Sigma-A5316; 1:10,000 dilution), MSK1 (Santacruz-9392; 1:2000 dilution), ph-MSK1 (Abcam-31190; 1:3000 dilution), ERK1/2 (Santacruz-292838; 1:2000 dilution), ph-ERK (Cell signaling-9910; 1:2000 dilution), p38 (Santacruz-728; 1:2000 dilution), ph-p38 (Cell signaling-9910; 1:2000 dilution), and anti-flag (Sigma-F3165; 1:5000 dilution).

    Techniques: Immunohistochemistry, Expressing, Immunofluorescence, Staining

    Schematic representation showing p38-MAPK/MSK1 governing upregulation of H3S10ph and MSK1 as a potential target in gastric cancer

    Journal: Clinical Epigenetics

    Article Title: p38-MAPK/MSK1-mediated overexpression of histone H3 serine 10 phosphorylation defines distance-dependent prognostic value of negative resection margin in gastric cancer

    doi: 10.1186/s13148-016-0255-9

    Figure Lengend Snippet: Schematic representation showing p38-MAPK/MSK1 governing upregulation of H3S10ph and MSK1 as a potential target in gastric cancer

    Article Snippet: Proteins on PVDF membrane were hybridized with anti-H3 (Upstate-06-755; 1:2000 dilution), H4 (Millipore-07-108; 1:4000 dilution), H3S10ph (Millipore-06-570; 1:7000 dilution), H4K16ac (Millipore-07-329; 1:8000 dilution), H4K20me3 (Abcam-9053; 1:4000 dilution), β-actin (Sigma-A5316; 1:10,000 dilution), MSK1 (Santacruz-9392; 1:2000 dilution), ph-MSK1 (Abcam-31190; 1:3000 dilution), ERK1/2 (Santacruz-292838; 1:2000 dilution), ph-ERK (Cell signaling-9910; 1:2000 dilution), p38 (Santacruz-728; 1:2000 dilution), ph-p38 (Cell signaling-9910; 1:2000 dilution), and anti-flag (Sigma-F3165; 1:5000 dilution).

    Techniques:

    The Ser-358 residue is required for hKOR activation of p38 MAPK. a , HEK293 cells expressing hKOR, hKOR(S358N), rKOR, or untransfected wild type HEK293 were treated 5–120 min with U50,488 (10 μ m ) before cell lysis and immunoblotted for

    Journal: The Journal of Biological Chemistry

    Article Title: Ligand Directed Signaling Differences between Rodent and Human ?-Opioid Receptors *

    doi: 10.1074/jbc.M112.381368

    Figure Lengend Snippet: The Ser-358 residue is required for hKOR activation of p38 MAPK. a , HEK293 cells expressing hKOR, hKOR(S358N), rKOR, or untransfected wild type HEK293 were treated 5–120 min with U50,488 (10 μ m ) before cell lysis and immunoblotted for

    Article Snippet: HEK293 cells stably expressing either rKOR or hKOR were treated for 30 min with either U50,488 or pentazocine at concentrations from 100 p m to 10 μ m before lysis, and phospho-p38-ir was measured by Western blot ( , ).

    Techniques: Activation Assay, Expressing, Lysis

    Pentazocine is less efficacious than U50,488 in activation of p38. Pentazocine, but not U50,488, is more potent for activation of the p38 pathway in hKOR as compared with rKOR. a and b , HEK293 cells expressing hKOR or rKOR were treated for 30 min with

    Journal: The Journal of Biological Chemistry

    Article Title: Ligand Directed Signaling Differences between Rodent and Human ?-Opioid Receptors *

    doi: 10.1074/jbc.M112.381368

    Figure Lengend Snippet: Pentazocine is less efficacious than U50,488 in activation of p38. Pentazocine, but not U50,488, is more potent for activation of the p38 pathway in hKOR as compared with rKOR. a and b , HEK293 cells expressing hKOR or rKOR were treated for 30 min with

    Article Snippet: HEK293 cells stably expressing either rKOR or hKOR were treated for 30 min with either U50,488 or pentazocine at concentrations from 100 p m to 10 μ m before lysis, and phospho-p38-ir was measured by Western blot ( , ).

    Techniques: Activation Assay, Expressing

    Arrestin-3 is required for hKOR induced p38 MAPK phosphorylation. a , HEK293 cells expressing hKOR were transiently transfected with siRNA against arrestin-2, arrestin-3, or scrambled siRNA control. After 72 h, cells were treated with U50,488 (10 μ

    Journal: The Journal of Biological Chemistry

    Article Title: Ligand Directed Signaling Differences between Rodent and Human ?-Opioid Receptors *

    doi: 10.1074/jbc.M112.381368

    Figure Lengend Snippet: Arrestin-3 is required for hKOR induced p38 MAPK phosphorylation. a , HEK293 cells expressing hKOR were transiently transfected with siRNA against arrestin-2, arrestin-3, or scrambled siRNA control. After 72 h, cells were treated with U50,488 (10 μ

    Article Snippet: HEK293 cells stably expressing either rKOR or hKOR were treated for 30 min with either U50,488 or pentazocine at concentrations from 100 p m to 10 μ m before lysis, and phospho-p38-ir was measured by Western blot ( , ).

    Techniques: Expressing, Transfection

    Do hKOR and rKOR Differ in Their Activation of p38 MAPK?

    Journal: The Journal of Biological Chemistry

    Article Title: Ligand Directed Signaling Differences between Rodent and Human ?-Opioid Receptors *

    doi: 10.1074/jbc.M112.381368

    Figure Lengend Snippet: Do hKOR and rKOR Differ in Their Activation of p38 MAPK?

    Article Snippet: HEK293 cells stably expressing either rKOR or hKOR were treated for 30 min with either U50,488 or pentazocine at concentrations from 100 p m to 10 μ m before lysis, and phospho-p38-ir was measured by Western blot ( , ).

    Techniques: Activation Assay

    Macrophages are activated by PLGA microspheres in vitro , while PK-p38 i treatment inhibits p38 activation (a) RAW 264.7 macrophages were treated with empty PCADK and PLGA particles for 2, 4, and 6 hours. Densitometric evaluation (mean + SEM; n=3) of Western blots (representative blot for phospho-p38 shown) demonstrate that PCADK did not elevate p38-phosphorylation at any time point. PLGA increased p38-phosphorylation in a time dependent manner, resulting in a four-fold increase in activation at 4 and 6 hours (*p

    Journal: Nature materials

    Article Title: Sustained release of a p38-inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction

    doi: 10.1038/nmat2299

    Figure Lengend Snippet: Macrophages are activated by PLGA microspheres in vitro , while PK-p38 i treatment inhibits p38 activation (a) RAW 264.7 macrophages were treated with empty PCADK and PLGA particles for 2, 4, and 6 hours. Densitometric evaluation (mean + SEM; n=3) of Western blots (representative blot for phospho-p38 shown) demonstrate that PCADK did not elevate p38-phosphorylation at any time point. PLGA increased p38-phosphorylation in a time dependent manner, resulting in a four-fold increase in activation at 4 and 6 hours (*p

    Article Snippet: Proteins were transferred to a PVDF membrane (Bio-Rad) and blots probed with an antibody for phospho-specific p38 (Cell Signaling).

    Techniques: In Vitro, Activation Assay, Western Blot

    PK-p38 i therapy results in improved cardiac function and reduced fibrosis (a) Dimensions of the left ventricle were taken at systole and diastole using MRI at day seven and day 21 post-occlusion. PK-p38 i showed a statistically significant difference between days 7 and 21, whereas all other treatments did not reach significance (mean + SEM, n > 4, *p

    Journal: Nature materials

    Article Title: Sustained release of a p38-inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction

    doi: 10.1038/nmat2299

    Figure Lengend Snippet: PK-p38 i therapy results in improved cardiac function and reduced fibrosis (a) Dimensions of the left ventricle were taken at systole and diastole using MRI at day seven and day 21 post-occlusion. PK-p38 i showed a statistically significant difference between days 7 and 21, whereas all other treatments did not reach significance (mean + SEM, n > 4, *p

    Article Snippet: Proteins were transferred to a PVDF membrane (Bio-Rad) and blots probed with an antibody for phospho-specific p38 (Cell Signaling).

    Techniques: Magnetic Resonance Imaging

    PK-p38 i particles inhibit p38 phosphorylation, superoxide production, and TNF-α production in vivo following infarction (a) PK-p38 i treatment inhibited phosphorylation of p38 at three and seven days in the infarct zone while free inhibitor (p38 i ) or empty particles had no effect on phosphorylation at either time point (mean ± SEM, n ≥ 4, *p

    Journal: Nature materials

    Article Title: Sustained release of a p38-inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction

    doi: 10.1038/nmat2299

    Figure Lengend Snippet: PK-p38 i particles inhibit p38 phosphorylation, superoxide production, and TNF-α production in vivo following infarction (a) PK-p38 i treatment inhibited phosphorylation of p38 at three and seven days in the infarct zone while free inhibitor (p38 i ) or empty particles had no effect on phosphorylation at either time point (mean ± SEM, n ≥ 4, *p

    Article Snippet: Proteins were transferred to a PVDF membrane (Bio-Rad) and blots probed with an antibody for phospho-specific p38 (Cell Signaling).

    Techniques: In Vivo

    IL-38 inhibits IL-36γ-induced P38 and NF-κB signalings, as well as regulates the expression of differentiation markers and inflammatory molecules in human keratinocytes. All experiments were performed on keratinocyte cultures ( n = 3 strains) undergoing terminal differentiation and stimulated or not with IL-36γ in presence or absence of the indicated doses of IL-38 or IL-36Ra. a Protein extracts were obtained from keratinocyte cultures stimulated for 24 h and subjected to WB analysis to detect P38 and P65 phosphorylation. Filters were probed with anti-P38 and -P65 Abs. b Keratinocyte cultures were analyzed for KRT1, KRT10, Loricrin and ΔNp63 expression by WB. a , b β-actin was used as loading control and DI ratio indicates the densitometric intensity of the indicated phosphorylated/unphosphorylated proteins shown in one representative of three different WB. c mRNA levels of CXCL8, CCL20, VEGF-A, IL-6, HBD-2, and LL-37 were detected by real-time PCR analysis in keratinocytes stimulated for 6 h and normalized for GAPDH mRNA levels. TNF-α treatment was used as positive control. d ICAM-1 expression was evaluated by flow cytometry analysis on keratinocytes stimulated for 24 h with IL-36γ (50 ng/ml) and shown as mean fluorescence intensity. All data shown are the mean of three different experiments. c * p ≤ 0.05 and p ** ≤ 0.01 compared with untreated or IL-36γ-treated cultures, as assessed by Mann–Whitney U test

    Journal: Cell Death & Disease

    Article Title: IL-38 has an anti-inflammatory action in psoriasis and its expression correlates with disease severity and therapeutic response to anti-IL-17A treatment

    doi: 10.1038/s41419-018-1143-3

    Figure Lengend Snippet: IL-38 inhibits IL-36γ-induced P38 and NF-κB signalings, as well as regulates the expression of differentiation markers and inflammatory molecules in human keratinocytes. All experiments were performed on keratinocyte cultures ( n = 3 strains) undergoing terminal differentiation and stimulated or not with IL-36γ in presence or absence of the indicated doses of IL-38 or IL-36Ra. a Protein extracts were obtained from keratinocyte cultures stimulated for 24 h and subjected to WB analysis to detect P38 and P65 phosphorylation. Filters were probed with anti-P38 and -P65 Abs. b Keratinocyte cultures were analyzed for KRT1, KRT10, Loricrin and ΔNp63 expression by WB. a , b β-actin was used as loading control and DI ratio indicates the densitometric intensity of the indicated phosphorylated/unphosphorylated proteins shown in one representative of three different WB. c mRNA levels of CXCL8, CCL20, VEGF-A, IL-6, HBD-2, and LL-37 were detected by real-time PCR analysis in keratinocytes stimulated for 6 h and normalized for GAPDH mRNA levels. TNF-α treatment was used as positive control. d ICAM-1 expression was evaluated by flow cytometry analysis on keratinocytes stimulated for 24 h with IL-36γ (50 ng/ml) and shown as mean fluorescence intensity. All data shown are the mean of three different experiments. c * p ≤ 0.05 and p ** ≤ 0.01 compared with untreated or IL-36γ-treated cultures, as assessed by Mann–Whitney U test

    Article Snippet: Anti-phospho-P65, anti-P65, anti-phospho-P38, and ant-P38 were from Cell Signaling Technology (Denvers, MA, USA), whereas anti-KRT1, anti-KRT10, and anti-loricrin were from Covance (Emeryville, CA, USA).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Positive Control, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    p38 activation is required for the stimulatory effect of adenosine on alternatively activated macrophages. A ) Adenosine increases p38 activation. Macrophages were treated with IL-4 in the presence or absence of adenosine (ado) for 20 min. p38 activation

    Journal: The FASEB Journal

    Article Title: Adenosine promotes alternative macrophage activation via A2A and A2B receptors

    doi: 10.1096/fj.11-190934

    Figure Lengend Snippet: p38 activation is required for the stimulatory effect of adenosine on alternatively activated macrophages. A ) Adenosine increases p38 activation. Macrophages were treated with IL-4 in the presence or absence of adenosine (ado) for 20 min. p38 activation

    Article Snippet: Phospho-p38, phospho-STAT-6, phospho-Janus kinase (Jak) 1/3, phospho-SHC-adaptor protein (Shc), phospho-phosphatidylinositol 3-kinase (PI3K), phospho-Akt, phospho-p42/44, phospho-c-jun terminal kinase (JNK), CREB and arginase-1 protein levels were analyzed using 20–30 μg of cellular extracts using antibodies raised against doubly phosphorylated phospho-p38 (Cell Signaling, Danvers, MA, USA), phospho-STAT6 (Abcam, Cambridge, MA, USA), doubly phosphorylated phospho-Jak1 (Cell Signaling), phospho-Jak3 (Abcam), phospho-Shc (Cell Signaling), phospho-Akt (Cell Signaling), phospho-PI3K (Cell Signaling), doubly phosphorylated phospho-p42/44 (Cell Signaling), doubly phosphorylated phospho-JNK (Cell Signaling), CREB (Cell Signaling), and arginase-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Activation Assay

    MiR-16 alleviates inflammatory pain by inhibition of p38 MAPK. ( A ) Relative p-p38 expression level after infection of CFA; ( B ) Mechanical withdrawal threshold after injection; ( C ) Thermal withdrawal latencies after injection; ( D ) Frequency responses to cold stimulation after injection. MiR – microRNA; CFA – complete Freund’s adjuvant; MAPK – mitogen-activated protein kinase; p-p38 – phosphorylation of p38; * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-16 Alleviates Inflammatory Pain by Targeting Ras-Related Protein 23 (RAB23) and Inhibiting p38 MAPK Activation

    doi: 10.12659/MSM.897580

    Figure Lengend Snippet: MiR-16 alleviates inflammatory pain by inhibition of p38 MAPK. ( A ) Relative p-p38 expression level after infection of CFA; ( B ) Mechanical withdrawal threshold after injection; ( C ) Thermal withdrawal latencies after injection; ( D ) Frequency responses to cold stimulation after injection. MiR – microRNA; CFA – complete Freund’s adjuvant; MAPK – mitogen-activated protein kinase; p-p38 – phosphorylation of p38; * P

    Article Snippet: Thereafter, the membranes were blocked with 5% nonfat milk and incubated overnight at 4°C with anti-phosphorylation-p38 (p-p38) antibody (1:500; Cell Signaling), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling).

    Techniques: Inhibition, Expressing, Infection, Injection

    NRG1 treatment to microglial cells induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. a and b : Addition of NRG1 (10 nM) to resting microglial cells induced the phosphorylation of ERK1/2 as assessed by Western Blots. A representative membrane for one experiment is shown in a . In b we show the time-course of ERK1 (black bars) and ERK2 (grey bars) phosphorylation after NRG1 treatment (ratio of phospho-ERK over total ERK). There was a significant increase in ERK1 and 2 phosphorylation after 60 min of NRG1 treatment compared with resting state or control (ERK1: control versus 60 min NRG1 treatment P = 0.02 one-way ANOVA on ranks, ERK2: control versus 60 min NRG1 treatment P = 0.003 one-way ANOVA on ranks, n = 4). c and d : In the same way we assessed Akt phosphorylation after NRG1 treatment. In (c) a representative membrane of one experiment is shown. In (d) we show the time-course of Akt phosphorylation (ratio of phospho-Akt over total Akt) after NRG1 treatment of three independent experiments. There was a significant increase in Akt phosphorylation after 60 min of NRG1 treatment compared with resting state or control ( P = 0.002, one-way ANOVA, Bonferroni post hoc test). e and f : NRG1 treatment in microglia did not elicit phosphorylation of p38MAPK ( P = 0.7, t -test, n = 3). In e we show the time-course after NRG1 treatment where no phosphorylation of p38MAPK was seen. We tested if NRG1 could enhance p38MAPK phosphorylation of LPS treated microglia but could not observe any increase in phospho-p38 when treating LPS primed microglia with NRG1. In (f) a representative membrane is shown, in (g) we show quantification of phospho-p38 over p38 of 3 independent experiments (NRG1 vs. LPS or LPS+NRG1 P = 0.003, LPS vs. LPS+NRG1 P = 0.5, one-way ANOVA, Bonferroni post hoc test, n = 3). In h – j we show that only the MEK inhibitor U0126 could block ERK1 and 2 phosphorylation (h) and phosphorylation of ERK remained the same when cells were treated with the PI3K inhibitor Wortmannin (i). Quantification of three independent experiments is shown in j (for ERK 1 and 2 the phospho/total ratio was significantly different between NRG1 treated cells compared with NRG1 plus U0126 P

    Journal: Glia

    Article Title: Following Nerve Injury Neuregulin-1 Drives Microglial Proliferation and Neuropathic Pain via the MEK/ERK Pathway

    doi: 10.1002/glia.21124

    Figure Lengend Snippet: NRG1 treatment to microglial cells induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. a and b : Addition of NRG1 (10 nM) to resting microglial cells induced the phosphorylation of ERK1/2 as assessed by Western Blots. A representative membrane for one experiment is shown in a . In b we show the time-course of ERK1 (black bars) and ERK2 (grey bars) phosphorylation after NRG1 treatment (ratio of phospho-ERK over total ERK). There was a significant increase in ERK1 and 2 phosphorylation after 60 min of NRG1 treatment compared with resting state or control (ERK1: control versus 60 min NRG1 treatment P = 0.02 one-way ANOVA on ranks, ERK2: control versus 60 min NRG1 treatment P = 0.003 one-way ANOVA on ranks, n = 4). c and d : In the same way we assessed Akt phosphorylation after NRG1 treatment. In (c) a representative membrane of one experiment is shown. In (d) we show the time-course of Akt phosphorylation (ratio of phospho-Akt over total Akt) after NRG1 treatment of three independent experiments. There was a significant increase in Akt phosphorylation after 60 min of NRG1 treatment compared with resting state or control ( P = 0.002, one-way ANOVA, Bonferroni post hoc test). e and f : NRG1 treatment in microglia did not elicit phosphorylation of p38MAPK ( P = 0.7, t -test, n = 3). In e we show the time-course after NRG1 treatment where no phosphorylation of p38MAPK was seen. We tested if NRG1 could enhance p38MAPK phosphorylation of LPS treated microglia but could not observe any increase in phospho-p38 when treating LPS primed microglia with NRG1. In (f) a representative membrane is shown, in (g) we show quantification of phospho-p38 over p38 of 3 independent experiments (NRG1 vs. LPS or LPS+NRG1 P = 0.003, LPS vs. LPS+NRG1 P = 0.5, one-way ANOVA, Bonferroni post hoc test, n = 3). In h – j we show that only the MEK inhibitor U0126 could block ERK1 and 2 phosphorylation (h) and phosphorylation of ERK remained the same when cells were treated with the PI3K inhibitor Wortmannin (i). Quantification of three independent experiments is shown in j (for ERK 1 and 2 the phospho/total ratio was significantly different between NRG1 treated cells compared with NRG1 plus U0126 P

    Article Snippet: Membranes were incubated with primary antibody, rabbit phospho-Akt (1:1,000), rabbit Akt (1:1,000), rabbit phospho-Erk (1:2,000), rabbit Erk (1:2,000), rabbit phospho-p38 (1:500), rabbit p38 (1:500), diluted in 5% BSA in TBS-T (TBS + 0.1% Tween 20, all from Cell Signalling) with gentle shaking at 4°C overnight.

    Techniques: Western Blot, Blocking Assay

    Conserved pathways facilitate Tdp1 entry into human and mouse mitochondria following H 2 O 2 stress. ( a ) Left panel: Immunoblot of human fibroblast mitochondrial lysates without or with knockdown of ERK1 and P38 and in the absence or presence of 1 µM H 2 O 2 for 1 hour. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. Tdp1 levels were normalized to complex IV and fold changes are shown relative to levels in cells treated with nontargeting siRNA. ( b ) Left panel: Immunoblot of Rho-zero MEF mitochondrial lysates without or with knockdown of ERK1 and P38 and in the absence or presence of H 2 O 2 treatment. Mitochondrial complex IV was used as a loading control. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. ( c ) Left panel: Immunoblot of human fibroblast mitochondrial lysates without or with knockdown of ERK1 and P38 and in the presence of H 2 O 2 treatment. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. ( d ) Upper panel: Immunofluorescent staining of P38 and ERK1 in human fibroblasts before and after 5 minutes with 1 µM H 2 O 2 or 200 nM rotenone treatment. Lower panel: Immunofluorescent staining of Tdp1 in human fibroblasts treated with non-targeting siRNA or TIM23 siRNA with or without 1 µM H 2 O 2 for 1 hour. ( e ) Upper panel: Immunoblot of lysates from purified mitochondria of cultured MEFs expressing wt or S81A HA-tagged Tdp1 cDNA in the presence or absence of treatment with 1 µM H 2 O 2 for 1 hour. Lower panel: Graph of fold change in mitochondrial Tdp1 across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. Tdp1 levels were normalized to complex IV levels. Error bars represent one standard deviation. * P

    Journal: Scientific Reports

    Article Title: Reactive oxygen species stress increases accumulation of tyrosyl-DNA phsosphodiesterase 1 within mitochondria

    doi: 10.1038/s41598-018-22547-8

    Figure Lengend Snippet: Conserved pathways facilitate Tdp1 entry into human and mouse mitochondria following H 2 O 2 stress. ( a ) Left panel: Immunoblot of human fibroblast mitochondrial lysates without or with knockdown of ERK1 and P38 and in the absence or presence of 1 µM H 2 O 2 for 1 hour. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. Tdp1 levels were normalized to complex IV and fold changes are shown relative to levels in cells treated with nontargeting siRNA. ( b ) Left panel: Immunoblot of Rho-zero MEF mitochondrial lysates without or with knockdown of ERK1 and P38 and in the absence or presence of H 2 O 2 treatment. Mitochondrial complex IV was used as a loading control. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. ( c ) Left panel: Immunoblot of human fibroblast mitochondrial lysates without or with knockdown of ERK1 and P38 and in the presence of H 2 O 2 treatment. Right panel: Graph of mitochondrial Tdp1 levels across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. ( d ) Upper panel: Immunofluorescent staining of P38 and ERK1 in human fibroblasts before and after 5 minutes with 1 µM H 2 O 2 or 200 nM rotenone treatment. Lower panel: Immunofluorescent staining of Tdp1 in human fibroblasts treated with non-targeting siRNA or TIM23 siRNA with or without 1 µM H 2 O 2 for 1 hour. ( e ) Upper panel: Immunoblot of lysates from purified mitochondria of cultured MEFs expressing wt or S81A HA-tagged Tdp1 cDNA in the presence or absence of treatment with 1 µM H 2 O 2 for 1 hour. Lower panel: Graph of fold change in mitochondrial Tdp1 across three independent experiments following treatment with 1 µM H 2 O 2 for 1 hour. Tdp1 levels were normalized to complex IV levels. Error bars represent one standard deviation. * P

    Article Snippet: Rho-zero MEFs were transfected with Erk1 siRNA (Thermo Scientific/Dharmacon; L-040126-00-0005) or p38 siRNA (Cell Signaling; 6417 S).

    Techniques: Staining, Purification, Cell Culture, Expressing, Standard Deviation

    A CaMKII inhibitor, KN93, suppresses GW7845-induced MAPK activation. BU-11 cells were pretreated with Vh (DMSO, 0.1%) or KN93 (1–5μM) for 30 min and then treated with Vh (ethanol:DMSO, 50:50, 0.1%) or GW7845 (40μM) for 30 min. Cytoplasmic extracts were prepared and analyzed for p38 MAPK activation by detection of phosphorylated p38 MAPK by immunoblotting (A), for JNK activation by in vitro kinase assay (B), and for endogenous p38 MAPK/JNK activity by detection of phosphorylated ATF-2 by immunoblotting (C). Protein expression was quantified as described in the Materials and Methods and presented as “Fold Change from Naive.” Data are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p

    Journal: Toxicological Sciences

    Article Title: The Role of CaMKII in Calcium-Activated Death Pathways in Bone Marrow B Cells

    doi: 10.1093/toxsci/kfq256

    Figure Lengend Snippet: A CaMKII inhibitor, KN93, suppresses GW7845-induced MAPK activation. BU-11 cells were pretreated with Vh (DMSO, 0.1%) or KN93 (1–5μM) for 30 min and then treated with Vh (ethanol:DMSO, 50:50, 0.1%) or GW7845 (40μM) for 30 min. Cytoplasmic extracts were prepared and analyzed for p38 MAPK activation by detection of phosphorylated p38 MAPK by immunoblotting (A), for JNK activation by in vitro kinase assay (B), and for endogenous p38 MAPK/JNK activity by detection of phosphorylated ATF-2 by immunoblotting (C). Protein expression was quantified as described in the Materials and Methods and presented as “Fold Change from Naive.” Data are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p

    Article Snippet: Antibodies specific for cleaved caspase-3, p38 MAPK, phospho-p38 MAPK, ATF-2, and phospho-ATF-2 were from Cell Signaling Technology (Beverly, MA).

    Techniques: Activation Assay, In Vitro, Kinase Assay, Activity Assay, Expressing

    The immunotoxicant TBT induces calcium-dependent cell death. BU-11 cells were treated with Vh (DMSO, 0.1%) or TBT (1μM) for 5 min 2 h. (A) Caspase-3 activity and LDH release were assessed using the Caspase3/7-Glo Assay and the CytoTox-Glo Assay (Promega). Luminescence values in experimental wells were normalized by that measured in untreated cells and are presented as “Fold Change from Naïve.” (B) Formation of cleaved active caspase-3 (17 kDa) was analyzed by immunoblotting of cytoplasmic extracts. Protein expression was quantified as described in the Materials and Methods and presented as “Relative Expression.” (C) Cytoplasmic Ca 2+ was detected by loading BU-11 cells with Fluo4-AM (1μM) prior to treatment with Vh or TBT for 15 min, followed by flow cytometry. (D) CaMKII activity was determined in cytoplasmic extracts using the SignaTect Assay (Promega). CPM values in experimental wells were normalized by that measured in untreated cells and are presented as “Fold Change from Naïve.” (E) p38 MAPK activation was determined by detection of phosphorylated p38 MAPK in cytoplasmic extracts by immunoblotting. (F) BU-11 cells were pretreated with Vh (DMSO, 0.1%) or AIP-II (4μM) for 30 min and then treated with Vh (DMSO, 0.1%) or TBT (1 μM). Caspase-3 activity was assessed using the Caspase3/7-Glo Assay, as described in (A). Data are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p

    Journal: Toxicological Sciences

    Article Title: The Role of CaMKII in Calcium-Activated Death Pathways in Bone Marrow B Cells

    doi: 10.1093/toxsci/kfq256

    Figure Lengend Snippet: The immunotoxicant TBT induces calcium-dependent cell death. BU-11 cells were treated with Vh (DMSO, 0.1%) or TBT (1μM) for 5 min 2 h. (A) Caspase-3 activity and LDH release were assessed using the Caspase3/7-Glo Assay and the CytoTox-Glo Assay (Promega). Luminescence values in experimental wells were normalized by that measured in untreated cells and are presented as “Fold Change from Naïve.” (B) Formation of cleaved active caspase-3 (17 kDa) was analyzed by immunoblotting of cytoplasmic extracts. Protein expression was quantified as described in the Materials and Methods and presented as “Relative Expression.” (C) Cytoplasmic Ca 2+ was detected by loading BU-11 cells with Fluo4-AM (1μM) prior to treatment with Vh or TBT for 15 min, followed by flow cytometry. (D) CaMKII activity was determined in cytoplasmic extracts using the SignaTect Assay (Promega). CPM values in experimental wells were normalized by that measured in untreated cells and are presented as “Fold Change from Naïve.” (E) p38 MAPK activation was determined by detection of phosphorylated p38 MAPK in cytoplasmic extracts by immunoblotting. (F) BU-11 cells were pretreated with Vh (DMSO, 0.1%) or AIP-II (4μM) for 30 min and then treated with Vh (DMSO, 0.1%) or TBT (1 μM). Caspase-3 activity was assessed using the Caspase3/7-Glo Assay, as described in (A). Data are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p

    Article Snippet: Antibodies specific for cleaved caspase-3, p38 MAPK, phospho-p38 MAPK, ATF-2, and phospho-ATF-2 were from Cell Signaling Technology (Beverly, MA).

    Techniques: Activity Assay, Glo Assay, Expressing, Flow Cytometry, Cytometry, Activation Assay

    GW7845 activates calcium-dependent MAPK activation in pro/pre-B cells. (A) Cytoplasmic Ca 2+ was detected by loading BU-11 cells with Fluo3-AM (1μM) prior to treatment with Vh (DMSO, 0.1%) or GW7845 (± 15μM BAPTA) for 15 min, followed by flow cytometry. (B–D) For MAPK activation, BU-11 cells were pretreated with Vh (DMSO, 0.1%) or BAPTA (5–15μM) for 30 min and then treated with Vh (ethanol:DMSO, 50:50, 0.1%) or GW7845 (40μM) for 30 min. Cytoplasmic extracts were prepared and analyzed for p38 MAPK activation by detection of phosphorylated p38 MAPK by immunoblotting (B), for JNK activation by in vitro kinase assay (C), and for endogenous p38 MAPK/JNK activity by detection of phosphorylated ATF-2 by immunoblotting (D). Protein expression was quantified as described in the Materials and Methods and presented as “Fold Change from Naive.” Data are representative of or are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p

    Journal: Toxicological Sciences

    Article Title: The Role of CaMKII in Calcium-Activated Death Pathways in Bone Marrow B Cells

    doi: 10.1093/toxsci/kfq256

    Figure Lengend Snippet: GW7845 activates calcium-dependent MAPK activation in pro/pre-B cells. (A) Cytoplasmic Ca 2+ was detected by loading BU-11 cells with Fluo3-AM (1μM) prior to treatment with Vh (DMSO, 0.1%) or GW7845 (± 15μM BAPTA) for 15 min, followed by flow cytometry. (B–D) For MAPK activation, BU-11 cells were pretreated with Vh (DMSO, 0.1%) or BAPTA (5–15μM) for 30 min and then treated with Vh (ethanol:DMSO, 50:50, 0.1%) or GW7845 (40μM) for 30 min. Cytoplasmic extracts were prepared and analyzed for p38 MAPK activation by detection of phosphorylated p38 MAPK by immunoblotting (B), for JNK activation by in vitro kinase assay (C), and for endogenous p38 MAPK/JNK activity by detection of phosphorylated ATF-2 by immunoblotting (D). Protein expression was quantified as described in the Materials and Methods and presented as “Fold Change from Naive.” Data are representative of or are presented as means ± SE from at least three independent experiments. *Statistically different from Vh-treated ( p

    Article Snippet: Antibodies specific for cleaved caspase-3, p38 MAPK, phospho-p38 MAPK, ATF-2, and phospho-ATF-2 were from Cell Signaling Technology (Beverly, MA).

    Techniques: Activation Assay, Flow Cytometry, Cytometry, In Vitro, Kinase Assay, Activity Assay, Expressing

    p38 and JNK phosphorylation in the CPu in D1, D3 receptor mutant, and wild-type mice is not obviously changed after acute cocaine treatment. Time course of cocaine-induced phosphorylation of p38 and JNK in the CPu in wild-type ( A ), D1 ( B ), and D3 receptor mutant ( C ) mice. The phosphorylation status of p38 and JNK was determined 10, 20, 60, and 120 min after a cocaine injection (30 mg/kg, i.p.). Total cell extracts were analyzed by Western blotting using antibodies against dually phosphorylated-p38 (Thr 180 and Tyr 182 ), dually phosphorylated-JNK (Thr 183 and Tyr 185 ), total p38, or JNK, respectively. The same set of mice used in probing ERK activation was used in analyzing p38 and JNK activation after cocaine injections.

    Journal: The Journal of Neuroscience

    Article Title: Cocaine-Induced Intracellular Signaling and Gene Expression Are Oppositely Regulated by the Dopamine D1 and D3 Receptors

    doi: 10.1523/JNEUROSCI.0060-04.2004

    Figure Lengend Snippet: p38 and JNK phosphorylation in the CPu in D1, D3 receptor mutant, and wild-type mice is not obviously changed after acute cocaine treatment. Time course of cocaine-induced phosphorylation of p38 and JNK in the CPu in wild-type ( A ), D1 ( B ), and D3 receptor mutant ( C ) mice. The phosphorylation status of p38 and JNK was determined 10, 20, 60, and 120 min after a cocaine injection (30 mg/kg, i.p.). Total cell extracts were analyzed by Western blotting using antibodies against dually phosphorylated-p38 (Thr 180 and Tyr 182 ), dually phosphorylated-JNK (Thr 183 and Tyr 185 ), total p38, or JNK, respectively. The same set of mice used in probing ERK activation was used in analyzing p38 and JNK activation after cocaine injections.

    Article Snippet: Antibodies against ERK, JNK, p38 and phospho-ERK, phospho-JNK, phospho-p38 (Cell Signaling Technology, Beverly, MA) and c-Fos, neogenin and synaptotagmin VII (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:1000 dilution, and antibodies against actin (Santa Cruz Biotechnology) were used at the 1:3000 dilution.

    Techniques: Mutagenesis, Mouse Assay, Injection, Western Blot, Activation Assay

    Effect of α-mangostin on MAPKs, PI3K/Akt, and cleaved caspase-3 (c.c-3) in INS-1 cells. ( A ) Protein expression levels of P-P38, P38, P-JNK, JNK, and GAPDH in INS-1 cells treated or untreated with STZ and 5 μM α-mangostin for 24 h. ( B ) Protein expression levels of P-PI3K, P-Akt, cleaved caspase-3, and GAPDH in INS-1 cells treated or untreated with STZ and 5 μM α-mangostin for 24 h. ( C ) Each bar graphs present the densitometric quantification results of Western blot bands. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Alpha-Mangostin Improves Insulin Secretion and Protects INS-1 Cells from Streptozotocin-Induced Damage

    doi: 10.3390/ijms19051484

    Figure Lengend Snippet: Effect of α-mangostin on MAPKs, PI3K/Akt, and cleaved caspase-3 (c.c-3) in INS-1 cells. ( A ) Protein expression levels of P-P38, P38, P-JNK, JNK, and GAPDH in INS-1 cells treated or untreated with STZ and 5 μM α-mangostin for 24 h. ( B ) Protein expression levels of P-PI3K, P-Akt, cleaved caspase-3, and GAPDH in INS-1 cells treated or untreated with STZ and 5 μM α-mangostin for 24 h. ( C ) Each bar graphs present the densitometric quantification results of Western blot bands. * p

    Article Snippet: The proteins (20 μg/lane) were electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel, blotted onto polyvinylidene difluoride membranes, and further incubated for 1 h with primary antibodies against phospho-insulin receptor (P-IR), phospho-insulin receptor substrate-1 (P-IRS-1 (Ser1101)), phospho-phosphatidylinositol-3 kinase (P-PI3K), phospho-Akt (P-Akt), phospho-ERK (P-ERK), pancreatic and duodenal homeobox 1 (Pdx1), phospho-p38 (P-p38), p38, phospho-c-Jun N-terminal kinase (P-JNK), JNK, cleaved caspase-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Boston, MA, USA) at room temperature.

    Techniques: Expressing, Western Blot

    Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

    Journal: Molecular Vision

    Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

    doi:

    Figure Lengend Snippet: Effect of MnTBAP on glucose-induced activation of Raf-1 and phosphorylation of p38 MAP kinase. Activation of Raf-1 and phosphorylation of p38 MAP kinase were determined by Western blot using β-actin as a loading standard. Each sample was run in duplicate, and the experiment was repeated with three or more cell preparations. The histogram represents the density of Raf-1 ( A ), or p-p38 ( B ) band that has been adjusted to the density of β-actin band in the same lane. The ratio obtained from 5 mM glucose is considered as 100%. Asterisk (*) represent p

    Article Snippet: Activation of p38 MAP kinase The activation of MAP kinase was determined by performing the Western blot analysis of the phosphorylation of p38 MAP kinase (a key signaling molecule for H-Ras-induced signaling pathway) using phospho-p38 (p-p38) antibodies from Cell Signaling Technology (Beverly, MA).

    Techniques: Activation Assay, Western Blot

    Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

    Journal: Molecular Vision

    Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

    doi:

    Figure Lengend Snippet: Effect of hydrogen peroxide on the activation of H-Ras and its signaling pathway. Bovine retinal endothelial cells were incubated with 250 μM H 2 O 2 for 1 h. At the end of the incubation, the cells were rinsed with DMEM, and incubated in 5 mM glucose and 20 mM glucose media for 96 h. The activation of H-Ras was determined by Raf-1 binding assay, Raf-1 by measuring the expression of Raf-1 by Western blot, and that of MAP kinase by quantifying the expression of phospho-p38 of MAP kinase. Each sample was analyzed in duplicate, and the Western blots presented are representative of at least 3-4 experiments.

    Article Snippet: Activation of p38 MAP kinase The activation of MAP kinase was determined by performing the Western blot analysis of the phosphorylation of p38 MAP kinase (a key signaling molecule for H-Ras-induced signaling pathway) using phospho-p38 (p-p38) antibodies from Cell Signaling Technology (Beverly, MA).

    Techniques: Activation Assay, Incubation, Binding Assay, Expressing, Western Blot

    Effect of MnSOD overexpression on diabetes-induced activation of retinal MAP kinase. Activation of MAP kinase was estimated by measuring the expression of phospho-p38 in the same retina as used for H-Ras ( A ). The retina samples were analyzed in duplicate. The expression of β-actin in each row was used to correct the expression of phospho-p38 MAP kinase. B : The histogram represents the ratio of the densities of p-p38 and β-actin in the same sample (quantified using Un-Scan-It gel software), and the values obtained from WT mice are considered as 100%. Asterisk (*) signifies p

    Journal: Molecular Vision

    Article Title: Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina

    doi:

    Figure Lengend Snippet: Effect of MnSOD overexpression on diabetes-induced activation of retinal MAP kinase. Activation of MAP kinase was estimated by measuring the expression of phospho-p38 in the same retina as used for H-Ras ( A ). The retina samples were analyzed in duplicate. The expression of β-actin in each row was used to correct the expression of phospho-p38 MAP kinase. B : The histogram represents the ratio of the densities of p-p38 and β-actin in the same sample (quantified using Un-Scan-It gel software), and the values obtained from WT mice are considered as 100%. Asterisk (*) signifies p

    Article Snippet: Activation of p38 MAP kinase The activation of MAP kinase was determined by performing the Western blot analysis of the phosphorylation of p38 MAP kinase (a key signaling molecule for H-Ras-induced signaling pathway) using phospho-p38 (p-p38) antibodies from Cell Signaling Technology (Beverly, MA).

    Techniques: Over Expression, Activation Assay, Expressing, Software, Mouse Assay

    Obese mice are more susceptible to UVB-induced phosphorylation of MAPK protein than wild-type mice. Chronic exposure of mice to UVB induces phosphorylation of ERK1/2 (A), JNK (B) and p38 (C) proteins of MAPK family, and induces inactivation of ERK1/2,

    Journal: Free radical biology & medicine

    Article Title: Obesity increases the risk of UV radiation-induced oxidative stress and activation of MAPK and NF-?B signaling

    doi: 10.1016/j.freeradbiomed.2006.10.049

    Figure Lengend Snippet: Obese mice are more susceptible to UVB-induced phosphorylation of MAPK protein than wild-type mice. Chronic exposure of mice to UVB induces phosphorylation of ERK1/2 (A), JNK (B) and p38 (C) proteins of MAPK family, and induces inactivation of ERK1/2,

    Article Snippet: Phosphorylated ERK1/2 (Thr202 /Tyr204 ), JNK (Thr183 /Tyr185 ), p38 (Thr180 /Tyr182 ) and non-phosphorylated ERK1/2, JNK and p38 antibodies and the anti-β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

    Techniques: Mouse Assay

    Effect of benidipine on ERK1/2 and p38 MAPK activity in ischemic-reperfused myocardial tissue. The ERK1/2 and p38 MAP kinase activity assays were performed by using ERK1/2 or p38 MAPK assay kits. EIk-1 fusion protein or ATF-2 fusion protein was used as substrate for ERK1/2 or p38 MAPK, respectively. # P

    Journal: British Journal of Pharmacology

    Article Title: Antiapoptotic mechanisms of benidipine in the ischemic/reperfused heart

    doi: 10.1038/sj.bjp.0705847

    Figure Lengend Snippet: Effect of benidipine on ERK1/2 and p38 MAPK activity in ischemic-reperfused myocardial tissue. The ERK1/2 and p38 MAP kinase activity assays were performed by using ERK1/2 or p38 MAPK assay kits. EIk-1 fusion protein or ATF-2 fusion protein was used as substrate for ERK1/2 or p38 MAPK, respectively. # P

    Article Snippet: The ERK1/2 and p38 MAP kinase activity assays were performed by using ERK1/2 or p38 MAPK assay kits (Cell Signaling Technology) according to the manufacturer's instructions.

    Techniques: Activity Assay

    IL-1β upregulates MMP2 and MMP9 expression and activity by activating p38. A : RT-PCR analysis showed that MMP2 and MMP9 mRNA were upregulated in both AGS and MKN-45 cells in response to treatment with IL-1β; this effect was blocked by p38 siRNA or the p38 inhibitor SB202190. B : Quantification of the expression of MMP2 and MMP9 mRNA normalized to GAPDH mRNA. ** P

    Journal: Molecular Cancer

    Article Title: IL-1?-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

    doi: 10.1186/1476-4598-13-18

    Figure Lengend Snippet: IL-1β upregulates MMP2 and MMP9 expression and activity by activating p38. A : RT-PCR analysis showed that MMP2 and MMP9 mRNA were upregulated in both AGS and MKN-45 cells in response to treatment with IL-1β; this effect was blocked by p38 siRNA or the p38 inhibitor SB202190. B : Quantification of the expression of MMP2 and MMP9 mRNA normalized to GAPDH mRNA. ** P

    Article Snippet: SiRNA against p38 (Cell Signaling), siRNA against JNK (Cell Signaling) or control siRNA (scrambled siRNA) (used as nonsilencing control) (Cell Signaling) and siRNA against MMP-2 or MMP-9 with the targeted position 498 and 2243 of human MMP-2, and targeted position 372 and 1312 of human MMP-9 which were exact the same as the introduction by Luo Y’s [ ] (Synthesized in GenePharma Company, Shanghai, China) were transfected into cells, respectively with Lipofectamine 2000 according to the manufacturer’s instructions.

    Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    IL-1β activates AP-1 activity through p38 pathway. A : Both GA cells were transfected with AP-1 reporter plasmid or AP-1 reporter plasmid together with scramble siRNA or p38 siRNA. AP-1 luciferase reporter gene activity was significantly increased by IL-1β stimulation in both AGS and MKN-45 cells; these effects were significantly inhibited by p38 siRNA in a dose-dependent manner and also inhibited by the p38 inhibitor SB202190. ** P

    Journal: Molecular Cancer

    Article Title: IL-1?-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

    doi: 10.1186/1476-4598-13-18

    Figure Lengend Snippet: IL-1β activates AP-1 activity through p38 pathway. A : Both GA cells were transfected with AP-1 reporter plasmid or AP-1 reporter plasmid together with scramble siRNA or p38 siRNA. AP-1 luciferase reporter gene activity was significantly increased by IL-1β stimulation in both AGS and MKN-45 cells; these effects were significantly inhibited by p38 siRNA in a dose-dependent manner and also inhibited by the p38 inhibitor SB202190. ** P

    Article Snippet: SiRNA against p38 (Cell Signaling), siRNA against JNK (Cell Signaling) or control siRNA (scrambled siRNA) (used as nonsilencing control) (Cell Signaling) and siRNA against MMP-2 or MMP-9 with the targeted position 498 and 2243 of human MMP-2, and targeted position 372 and 1312 of human MMP-9 which were exact the same as the introduction by Luo Y’s [ ] (Synthesized in GenePharma Company, Shanghai, China) were transfected into cells, respectively with Lipofectamine 2000 according to the manufacturer’s instructions.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase

    IL-1β promotes GA cell migration and invasion by activating p38. A : Western blots confirmed that p-p38 could be induced by 30 min stimulation with IL-1β in AGS and MKN-45 cell lines; the activation of p38 by IL-1β was inhibited by p38 inhibitor SB202190. B : Transfection of p38 siRNA knocked down p38 expression in both the two GA cell lines. C-F : Treatment of GA cells with IL-1β increased cell migration and invasion in vitro; these effects were inhibited by p38 siRNA or the p38 pathway inhibitor SB202190. ** P

    Journal: Molecular Cancer

    Article Title: IL-1?-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

    doi: 10.1186/1476-4598-13-18

    Figure Lengend Snippet: IL-1β promotes GA cell migration and invasion by activating p38. A : Western blots confirmed that p-p38 could be induced by 30 min stimulation with IL-1β in AGS and MKN-45 cell lines; the activation of p38 by IL-1β was inhibited by p38 inhibitor SB202190. B : Transfection of p38 siRNA knocked down p38 expression in both the two GA cell lines. C-F : Treatment of GA cells with IL-1β increased cell migration and invasion in vitro; these effects were inhibited by p38 siRNA or the p38 pathway inhibitor SB202190. ** P

    Article Snippet: SiRNA against p38 (Cell Signaling), siRNA against JNK (Cell Signaling) or control siRNA (scrambled siRNA) (used as nonsilencing control) (Cell Signaling) and siRNA against MMP-2 or MMP-9 with the targeted position 498 and 2243 of human MMP-2, and targeted position 372 and 1312 of human MMP-9 which were exact the same as the introduction by Luo Y’s [ ] (Synthesized in GenePharma Company, Shanghai, China) were transfected into cells, respectively with Lipofectamine 2000 according to the manufacturer’s instructions.

    Techniques: Migration, Western Blot, Activation Assay, Transfection, Expressing, In Vitro

    PD-mediated phosphorylation of proinflammatory p38 and JNK1/2 is reduced by chemical or genetic ablation of sEH. Phosphorylation and activation of p38 and JNK1/2 were quantified from all groups by normalizing band intensity to that of α -tubulin. (A) Original blots displaying two randomly selected animals. (B and C). Bar graphs of phosphorylation status of p38 and JNK1/2. Mean band intensity is measured for all six mice and is represented as arbitrary units (mean ± S.E.M.) (* P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title:

    doi: 10.1124/jpet.116.238113

    Figure Lengend Snippet: PD-mediated phosphorylation of proinflammatory p38 and JNK1/2 is reduced by chemical or genetic ablation of sEH. Phosphorylation and activation of p38 and JNK1/2 were quantified from all groups by normalizing band intensity to that of α -tubulin. (A) Original blots displaying two randomly selected animals. (B and C). Bar graphs of phosphorylation status of p38 and JNK1/2. Mean band intensity is measured for all six mice and is represented as arbitrary units (mean ± S.E.M.) (* P

    Article Snippet: Immunoblotting of lysates was performed with antibodies for monocyte chemotactic protein 1 (MCP-1; BioLegend, San Diego, CA), epidermal growth factor–like module-containing mucin-like hormone receptor-like 1 (F4/80), binding immunoglobulin protein, phosphorylated p38-pp38 (Thr180 /Tyr182 ), p38 mitogen-activated protein kinases (p38), phosphorylated c-Jun N-terminal kinase (JNK; Thr183 /Tyr185 ), JNK or cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), or α -tubulin (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Activation Assay, Mouse Assay

    ERK and p38 play opposite roles in regulating monocyte migration induced by LPS and MCP-1. A. WT monocytes were pre-treated with p38 inhibitor SB203580 (SB; 10 μM), ERK inhibitor PD98059 (PD; 50) or JNK inhibitor SP600125 (SP; 10 μM) for

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TLR4 Signaling Augments Monocyte Chemotaxis by Regulating G Protein-Coupled Receptor Kinase 2 (GRK2) Translocation

    doi: 10.4049/jimmunol.1300790

    Figure Lengend Snippet: ERK and p38 play opposite roles in regulating monocyte migration induced by LPS and MCP-1. A. WT monocytes were pre-treated with p38 inhibitor SB203580 (SB; 10 μM), ERK inhibitor PD98059 (PD; 50) or JNK inhibitor SP600125 (SP; 10 μM) for

    Article Snippet: In some cases, monocytes were preincubated with a variety of MAPK inhibitors: p38 inhibitor SB203580 (10 μM, Cell Signaling Technology, Boston, MA); ERK inhibitor PD98059 (50 μM, Cell Signaling Technology, Boston, MA) ( ); or JNK inhibitor SP600125 (10 μM, Sigma-Aldrich, St. Louis, MO) for 30 min ( ).

    Techniques: Migration

    Keratinocyte ligation to fibrillar type I collagen induced phosphorylation of ERK and p38 MAPK but not JNK Primary keratinocytes were plated on fibrillar type I collagen, and cell lysates were harvested at the designated time points. The 0-min cells were keratinocytes sampled before plating on collagen. Phosphorylated (p-) and total ERK, p38, and JNK were visualized by immunoblot using specific antibodies. Results are representative of three independent experiments with keratinocytes from 3 different batches of cells.

    Journal: The Journal of investigative dermatology

    Article Title: Cdc42 Inhibits ERK-mediated Collagenase-1 (MMP-1) Expression in Collagen-Activated Human Keratinocytes

    doi: 10.1038/jid.2013.499

    Figure Lengend Snippet: Keratinocyte ligation to fibrillar type I collagen induced phosphorylation of ERK and p38 MAPK but not JNK Primary keratinocytes were plated on fibrillar type I collagen, and cell lysates were harvested at the designated time points. The 0-min cells were keratinocytes sampled before plating on collagen. Phosphorylated (p-) and total ERK, p38, and JNK were visualized by immunoblot using specific antibodies. Results are representative of three independent experiments with keratinocytes from 3 different batches of cells.

    Article Snippet: Phospho-p44/42 MAPK XPTM rabbit monoclonal antibody, p44/42 MAPK rabbit monoclonal antibody, Phosphoplus® p38 MAPK antibody, Phosphoplus® SAPK/JNK antibody, and the rabbit monoclonal antibodies for RhoA (67B9) and Cdc42 (11A11) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Ligation