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  • 99
    Millipore jnk inhibitor sp600125
    AdoR promotes transcription of TNF Egr in polarity-deficient epithelial cells. a sal > ent2 , AdoR expression enhances TNF egr transcript levels in the wing disc independently of <t>JNK</t> signalling. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n = 6. b – d sal > ent2, AdoR expression upregulates expression of Egr-GFP from a genomic fosmid ( b , c ). This effect is not rescued by co-expression of JNK bskDN ( d ). The arrowhead in b indicates a suspected position effect of the attP site (VK00033) where the fosmid was integrated. e CADO treatment increases expression of TNF egr in explanted wild-type discs, but not in AdoR mutant discs. This effect is further enhanced by Ador overexpression. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n ≥ 5. f en > scrib Ri discs upregulate TNF egr mRNA in an AdoR/Ent2-dependent and JNK-independent fashion. Fold changes are relative to rp49 , n ≥ 4. g Human HaCaT cells treated with adenosine upregulates TNF-α transcript levels (as assayed by qRT-PCR). Fold changes are relative to GAPDH , n = 5. Expression of PKG1 , a housekeeping gene and survivin , a JNK target gene, are also shown. h The effect shown in g was suppressed by three different adenosine receptor antagonists (CGS15943, SCH58261 or Caffeine) and by a PKA inhibitor cocktail (Merck # 20-114), but not by JNK inhibitor <t>SP600125.</t> Fold changes are relative to GAPDH , n ≥ 3 and expression of PKG1 is shown. i , j AdoR mutants live longer than wild-type flies when continuously fed with a low dose of Bleomycin ( i ) but live shorter than wild-type flies during acute desiccation (dry starvation, j ). **** P
    Jnk Inhibitor Sp600125, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho jnk
    Dabrafenib reduced the expression of phosphorylated <t>JNK</t> and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of <t>ERK</t> inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value
    Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc jnk
    Dabrafenib reduced the expression of phosphorylated <t>JNK</t> and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of <t>ERK</t> inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value
    Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 13435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/jnk/product/Cell Signaling Technology Inc
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    99
    Cell Signaling Technology Inc p jnk
    Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, <t>p-JNK</t> and <t>p-P38,</t> were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.
    P Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti phospho jnk
    Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, <t>p-JNK</t> and <t>p-P38,</t> were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.
    Anti Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc sapk jnk
    Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, <t>p-JNK</t> and <t>p-P38,</t> were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.
    Sapk Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 2080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti p jnk
    The inhibition of <t>JNK</t> and Nod1 reduced the expression of <t>TNFα</t> and IL-1β in HAPI cells treated with LPS. HAPI cells were pretreated with SP600125 (SP, JNK inhibitor) for 1 h, then treated with LPS. SP600125 treatment reduced the expression of TNFα (A), and IL-1β (B), ** p
    Anti P Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology jnk1
    Effect of muscle-specific <t>JNK1</t> deficiency on hepatic lipogenic gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice was measured by quantitative RT-PCR analysis of mRNA. Data for the expression of transcription factors ( C/ebp α, C/ebp β, Ppar γ, and Srebp1 ), coactivators ( Pgc1 α and Pgc1 β), fatty acid synthase ( Fas ), and microsomal triglyceride transfer protein ( Mttp ) mRNA are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P
    Jnk1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1033 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti jnk
    PR55γ Is a Regulator of <t>JNK</t> following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. <t>GFP</t> expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002
    Anti Jnk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc c jun n terminal kinase jnk
    EGCG pretreatment of promyelocytic HL-60 inhibits S1P response in macrophage-differentiated HL-60 cells. Notes: Three different conditions were used to evaluate the EGCG impact on the S1P response. Predifferentiation conditions: serum-starved HL-60 cells were treated for 24 hours with various concentrations of EGCG (0–10 μM), then treated for 18 hours with 3 μM PMA, to induce HL-60 cell differentiation and adhesion ( A – C ). Codifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with either 3 μM PMA alone or with a combination of 3 μM PMA and various EGCG concentrations ( D – F ). Postdifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA, then adherent cells were treated for 24 hours with various EGCG concentrations ( G – I ). In all three conditions, adherent cells were then treated with 1 μM S1P for 30 seconds, then cell lysates were isolated, electrophoresed via SDS-PAGE, and immunodetection performed. Scanning densitometry of the autoradiograms was performed, and ratios of P-ERK/ERK, <t>P-JNK/JNK,</t> and P-P38/P38 were obtained. The data shown is representative of three independent experiments. Abbreviations: EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HL, human leukemia; P-P38, phosphorylated P38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; PMA, phorbol-12-myristate-13-acetate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; S1P, sphingosine-1-phosphate.
    C Jun N Terminal Kinase Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc total jnk
    EGCG pretreatment of promyelocytic HL-60 inhibits S1P response in macrophage-differentiated HL-60 cells. Notes: Three different conditions were used to evaluate the EGCG impact on the S1P response. Predifferentiation conditions: serum-starved HL-60 cells were treated for 24 hours with various concentrations of EGCG (0–10 μM), then treated for 18 hours with 3 μM PMA, to induce HL-60 cell differentiation and adhesion ( A – C ). Codifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with either 3 μM PMA alone or with a combination of 3 μM PMA and various EGCG concentrations ( D – F ). Postdifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA, then adherent cells were treated for 24 hours with various EGCG concentrations ( G – I ). In all three conditions, adherent cells were then treated with 1 μM S1P for 30 seconds, then cell lysates were isolated, electrophoresed via SDS-PAGE, and immunodetection performed. Scanning densitometry of the autoradiograms was performed, and ratios of P-ERK/ERK, <t>P-JNK/JNK,</t> and P-P38/P38 were obtained. The data shown is representative of three independent experiments. Abbreviations: EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HL, human leukemia; P-P38, phosphorylated P38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; PMA, phorbol-12-myristate-13-acetate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; S1P, sphingosine-1-phosphate.
    Total Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AdoR promotes transcription of TNF Egr in polarity-deficient epithelial cells. a sal > ent2 , AdoR expression enhances TNF egr transcript levels in the wing disc independently of JNK signalling. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n = 6. b – d sal > ent2, AdoR expression upregulates expression of Egr-GFP from a genomic fosmid ( b , c ). This effect is not rescued by co-expression of JNK bskDN ( d ). The arrowhead in b indicates a suspected position effect of the attP site (VK00033) where the fosmid was integrated. e CADO treatment increases expression of TNF egr in explanted wild-type discs, but not in AdoR mutant discs. This effect is further enhanced by Ador overexpression. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n ≥ 5. f en > scrib Ri discs upregulate TNF egr mRNA in an AdoR/Ent2-dependent and JNK-independent fashion. Fold changes are relative to rp49 , n ≥ 4. g Human HaCaT cells treated with adenosine upregulates TNF-α transcript levels (as assayed by qRT-PCR). Fold changes are relative to GAPDH , n = 5. Expression of PKG1 , a housekeeping gene and survivin , a JNK target gene, are also shown. h The effect shown in g was suppressed by three different adenosine receptor antagonists (CGS15943, SCH58261 or Caffeine) and by a PKA inhibitor cocktail (Merck # 20-114), but not by JNK inhibitor SP600125. Fold changes are relative to GAPDH , n ≥ 3 and expression of PKG1 is shown. i , j AdoR mutants live longer than wild-type flies when continuously fed with a low dose of Bleomycin ( i ) but live shorter than wild-type flies during acute desiccation (dry starvation, j ). **** P

    Journal: Nature Communications

    Article Title: Epithelial cells release adenosine to promote local TNF production in response to polarity disruption

    doi: 10.1038/s41467-018-07114-z

    Figure Lengend Snippet: AdoR promotes transcription of TNF Egr in polarity-deficient epithelial cells. a sal > ent2 , AdoR expression enhances TNF egr transcript levels in the wing disc independently of JNK signalling. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n = 6. b – d sal > ent2, AdoR expression upregulates expression of Egr-GFP from a genomic fosmid ( b , c ). This effect is not rescued by co-expression of JNK bskDN ( d ). The arrowhead in b indicates a suspected position effect of the attP site (VK00033) where the fosmid was integrated. e CADO treatment increases expression of TNF egr in explanted wild-type discs, but not in AdoR mutant discs. This effect is further enhanced by Ador overexpression. TNFR grnd expression, measured by RT-qPCR, is shown as a control. Fold changes are relative to rp49 , n ≥ 5. f en > scrib Ri discs upregulate TNF egr mRNA in an AdoR/Ent2-dependent and JNK-independent fashion. Fold changes are relative to rp49 , n ≥ 4. g Human HaCaT cells treated with adenosine upregulates TNF-α transcript levels (as assayed by qRT-PCR). Fold changes are relative to GAPDH , n = 5. Expression of PKG1 , a housekeeping gene and survivin , a JNK target gene, are also shown. h The effect shown in g was suppressed by three different adenosine receptor antagonists (CGS15943, SCH58261 or Caffeine) and by a PKA inhibitor cocktail (Merck # 20-114), but not by JNK inhibitor SP600125. Fold changes are relative to GAPDH , n ≥ 3 and expression of PKG1 is shown. i , j AdoR mutants live longer than wild-type flies when continuously fed with a low dose of Bleomycin ( i ) but live shorter than wild-type flies during acute desiccation (dry starvation, j ). **** P

    Article Snippet: Adenosine, SCH58261, Caffeine and the JNK inhibitor SP600125 were obtained from Sigma.

    Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Over Expression

    TRBP is phosphorylated by ERK and JNK in response to arsenite-induced oxidative stress. ( A ) TRBP’s electrophoretic mobility shifts in response to sodium arsenite treatment. A western blot analysis of 50 μg protein per lane from HeLa-TRBP cells treated with 10 μM sodium arsenite at the indicated time points is shown. Western blot analysis was performed with anti-Flag and anti-β actin antibodies. The slower migrating TRBP band at 8 h, and 12 h is indicated by an asterisk. The line between lanes 1 and 2 as well as between lanes 3 and 4 represents where lanes from the same western blot were joined. ( B ) PKR phosphorylation and eIF2α phosphorylation kinetics in response to sodium arsenite treatment. HeLa cells were treated with 10 µM sodium arsenite and cell extracts were prepared at the indicated time points. PKR and eIF2α phosphorylation status at each time point was determined by a western blot analysis using anti-phospho-PKR and anti-phospho-eIF2α specific antibodies using 100 µg and 10 µg of total protein respectively. Each blot was subsequently stripped and re-probed with anti- eIF2α or anti-PKR antibody to ensure equal loading in all lanes. ( C ) TRBP is phosphorylated in response to oxidative stress. Extracts from untreated, and 8 h, or 12 h arsenite-treated TRBP-HeLa cells were prepared in the presence or absence of phosphatase inhibitor (PPi) as indicated above the lanes and subsequently treated with phosphatase (Ptase) or left untreated as indicated. Western blot to was performed with anti-Flag antibody followed by anti- β-actin antibody. ( D ) ERK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor PD0325901 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-ERK, anti-total ERK, and anti-GAPDH antibodies. ( D ) JNK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor SP600125 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-JNK, anti-total JNK, and anti-GAPDH antibodies.

    Journal: Scientific Reports

    Article Title: Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

    doi: 10.1038/s41598-018-19360-8

    Figure Lengend Snippet: TRBP is phosphorylated by ERK and JNK in response to arsenite-induced oxidative stress. ( A ) TRBP’s electrophoretic mobility shifts in response to sodium arsenite treatment. A western blot analysis of 50 μg protein per lane from HeLa-TRBP cells treated with 10 μM sodium arsenite at the indicated time points is shown. Western blot analysis was performed with anti-Flag and anti-β actin antibodies. The slower migrating TRBP band at 8 h, and 12 h is indicated by an asterisk. The line between lanes 1 and 2 as well as between lanes 3 and 4 represents where lanes from the same western blot were joined. ( B ) PKR phosphorylation and eIF2α phosphorylation kinetics in response to sodium arsenite treatment. HeLa cells were treated with 10 µM sodium arsenite and cell extracts were prepared at the indicated time points. PKR and eIF2α phosphorylation status at each time point was determined by a western blot analysis using anti-phospho-PKR and anti-phospho-eIF2α specific antibodies using 100 µg and 10 µg of total protein respectively. Each blot was subsequently stripped and re-probed with anti- eIF2α or anti-PKR antibody to ensure equal loading in all lanes. ( C ) TRBP is phosphorylated in response to oxidative stress. Extracts from untreated, and 8 h, or 12 h arsenite-treated TRBP-HeLa cells were prepared in the presence or absence of phosphatase inhibitor (PPi) as indicated above the lanes and subsequently treated with phosphatase (Ptase) or left untreated as indicated. Western blot to was performed with anti-Flag antibody followed by anti- β-actin antibody. ( D ) ERK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor PD0325901 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-ERK, anti-total ERK, and anti-GAPDH antibodies. ( D ) JNK is phosphorylated in response to oxidative stress and phosphorylates TRBP. TRBP overexpressing TRBP-HeLa cells were treated with 10 μM arsenite alone or in combination with the MEK inhibitor SP600125 for 24 hours. Cell extracts were made at the indicated time points, and western blot analysis was performed using anti-Flag, anti-phospho-JNK, anti-total JNK, and anti-GAPDH antibodies.

    Article Snippet: Sodium arsenite, phosphatase inhibitor cocktail (Phosphatase Inhibitor Cocktail 2 – P5726), and the JNK inhibitor (SP600125, Catalog number S5567) were purchased from Sigma Aldrich.

    Techniques: Western Blot

    Inhibition of phosphorylation of MAPK blocks CAPE-enhanced expression of NDRG1 in TW01 cells. ( A )The expression levels of ERK, p-ERK, NDRG1, or β-actin after pretreatments with (+) or without (−) PD0325901 for 1 h before 30 μM CAPE treatment in TW01 cells were determined by immunoblotting and RT-qPCR ( B ) assays. TW01 cells were pretreated with (+) or without (−) SP600125 ( C ) or SB202190 ( D ). The protein levels of JNK, p-JNK, p38, p-p38, NDRG1, or β-actin in CAPE-treated TW01 cells were determined by immunoblotting assay. (** p

    Journal: International Journal of Molecular Sciences

    Article Title: Caffeic Acid Phenethyl Ester Induces N-myc Downstream Regulated Gene 1 to Inhibit Cell Proliferation and Invasion of Human Nasopharyngeal Cancer Cells

    doi: 10.3390/ijms19051397

    Figure Lengend Snippet: Inhibition of phosphorylation of MAPK blocks CAPE-enhanced expression of NDRG1 in TW01 cells. ( A )The expression levels of ERK, p-ERK, NDRG1, or β-actin after pretreatments with (+) or without (−) PD0325901 for 1 h before 30 μM CAPE treatment in TW01 cells were determined by immunoblotting and RT-qPCR ( B ) assays. TW01 cells were pretreated with (+) or without (−) SP600125 ( C ) or SB202190 ( D ). The protein levels of JNK, p-JNK, p38, p-p38, NDRG1, or β-actin in CAPE-treated TW01 cells were determined by immunoblotting assay. (** p

    Article Snippet: JNK inhibitor II (SP600125) was purchased from Millipore (Temecula, CA, USA).

    Techniques: Inhibition, Expressing, Quantitative RT-PCR

    Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value

    Journal: Human Molecular Genetics

    Article Title: In silico drug screening by using genome-wide association study data repurposed dabrafenib, an anti-melanoma drug, for Parkinson’s disease

    doi: 10.1093/hmg/ddy279

    Figure Lengend Snippet: Dabrafenib reduced the expression of phosphorylated JNK and c-jun in both SH-SY5Y cells and mice ( A ) LDH/cell viability assay under the condition of ERK inhibitor. Neuroprotective effects of dabrafenib (5 μM) were not dependent on the downregulation of activated ERK expression by PD98059 (30 μM) or U0126 (3 μM). Neuroprotective effects were assessed by the LDH and cell viability assay in SH-SY5Y cells at 24 h after MPP + (3 mM) exposure. ** , P -value

    Article Snippet: Antibodies against caspase-3 (#9665, 1:1000), cleaved caspase-9 (#7237, 1:1000), ERK 1/2 (#4695, 1:1000), phospho-ERK 1/2 (#4370, 1:2000), JNK (#9252, 1:1000), phospho-JNK (#4668, 1:1000), phospho-c-jun (#3270, 1:1000) and phospho-B-Raf (Ser445; #2696, 1:1000) were all purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Mouse Assay, Viability Assay

    Analyses of phosphorylation of MAPKs, TGF-β1 and Notch3 expression in lungs of (BLM) bleomycin-injured and control mice A. Representative images of H E staining and TGF-β1 expression determined by immunohistochemistry. Bar size, 100 μm. B. Western blot analyses of p38, JNK1/2 and ERK1/2 phosphorylation, α-SMA and Notch3 expression. The protein bands were quantified, normalized with their non-phosphorylated counterparts or β-actin, and presented relative to that of saline-treated controls as means ± SEM (n = 3-4). *, P

    Journal: Oncotarget

    Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

    doi: 10.18632/oncotarget.9667

    Figure Lengend Snippet: Analyses of phosphorylation of MAPKs, TGF-β1 and Notch3 expression in lungs of (BLM) bleomycin-injured and control mice A. Representative images of H E staining and TGF-β1 expression determined by immunohistochemistry. Bar size, 100 μm. B. Western blot analyses of p38, JNK1/2 and ERK1/2 phosphorylation, α-SMA and Notch3 expression. The protein bands were quantified, normalized with their non-phosphorylated counterparts or β-actin, and presented relative to that of saline-treated controls as means ± SEM (n = 3-4). *, P

    Article Snippet: Western analysis was performed as previously described [ ] by using antibodies against Notch3 (sc-5593), α-SMA and pro-collagen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (Sigma), and p-JNK1/2, p-p38, p-ERK1/2, JNK1/2, p38, ERK1/2, and β-actin (Cell signaling), and a chemiluminescence detection kit (LumiGLO® Reagent and Peroxide, Cell signaling).

    Techniques: Expressing, Mouse Assay, Staining, Immunohistochemistry, Western Blot

    Representative images of immunohistochemical analyses of p38, JNK1/2, and ERK1/2 phosphorylation, α-SMA, TGF-β1 and Notch3 proteins and H E staining in paraffin-embedded lung sections from patients with idiopathic pulmonary fibrosis (IPF) and normal lung parenchyma Bar szie, 100 μm.

    Journal: Oncotarget

    Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

    doi: 10.18632/oncotarget.9667

    Figure Lengend Snippet: Representative images of immunohistochemical analyses of p38, JNK1/2, and ERK1/2 phosphorylation, α-SMA, TGF-β1 and Notch3 proteins and H E staining in paraffin-embedded lung sections from patients with idiopathic pulmonary fibrosis (IPF) and normal lung parenchyma Bar szie, 100 μm.

    Article Snippet: Western analysis was performed as previously described [ ] by using antibodies against Notch3 (sc-5593), α-SMA and pro-collagen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (Sigma), and p-JNK1/2, p-p38, p-ERK1/2, JNK1/2, p38, ERK1/2, and β-actin (Cell signaling), and a chemiluminescence detection kit (LumiGLO® Reagent and Peroxide, Cell signaling).

    Techniques: Immunohistochemistry, Staining

    Schematic illustration of roles of ERK1/2, p38, JNK1/2 and Notch3 in basal and TGF-β1-induced myofibroblast differentiation ECM, extracellular matrix; ROS, reactive oxygen species; →, lead to/activate; , inhibit. Line thickness indicates the extent of impact. Superscripts indicate the level or activation status.

    Journal: Oncotarget

    Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

    doi: 10.18632/oncotarget.9667

    Figure Lengend Snippet: Schematic illustration of roles of ERK1/2, p38, JNK1/2 and Notch3 in basal and TGF-β1-induced myofibroblast differentiation ECM, extracellular matrix; ROS, reactive oxygen species; →, lead to/activate; , inhibit. Line thickness indicates the extent of impact. Superscripts indicate the level or activation status.

    Article Snippet: Western analysis was performed as previously described [ ] by using antibodies against Notch3 (sc-5593), α-SMA and pro-collagen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (Sigma), and p-JNK1/2, p-p38, p-ERK1/2, JNK1/2, p38, ERK1/2, and β-actin (Cell signaling), and a chemiluminescence detection kit (LumiGLO® Reagent and Peroxide, Cell signaling).

    Techniques: Activation Assay

    Analyses of the effect of p38, JNK1/2 and Notch3 inhibitions on TGF-β1-induced α-SMA expression and the regulation between MAPKs and Notch3 signaling in IMR-90 fibroblasts Cells were serum-starved overnight prior to treatment with TGF-β1 (200 pM). A-B. Effect of pretreatment with p38 inhibitor SB203580 (10 μM, 1 h) and JNK inhibitor SP600125 (20 μM, 1 h) on p38 and JNK1/2 phosphorylation and α-SMA level after TGF-β1 treatment. C. Impact of Notch3 siRNA knockdown or inhibitor DAPT (10 μM, 1 h) on TGF-β1-induced α-SMA expression. Cells were transfected with a scrambled control (siCtrl) or Notch3 siRNA (siNotch3) for 24 h prior to treatment with TGF-β1 for 48 h. D. Effect of DAPT pretreatment (10 μM, 1 h) on TGF-β1 (1 h)-induced p38 and JNK1/2 activation, as well as ERK1/2 activity. E. Effect of p38 inhibitor SB203580 (10 μM, 1 h), JNK inhibitor SP600125 (20 μM, 1 h) and ERK inhibitor U0126 (10 μM, 1 h) on Notch3 expression after TGF-β1 treatment for 48 h. The protein levels of α-SMA and Notch3 were quantified, normalized by β-actin and presented relative to the one treated with TGF-β1 alone (100%) as means ± SEM (n = 3-4). *, P

    Journal: Oncotarget

    Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

    doi: 10.18632/oncotarget.9667

    Figure Lengend Snippet: Analyses of the effect of p38, JNK1/2 and Notch3 inhibitions on TGF-β1-induced α-SMA expression and the regulation between MAPKs and Notch3 signaling in IMR-90 fibroblasts Cells were serum-starved overnight prior to treatment with TGF-β1 (200 pM). A-B. Effect of pretreatment with p38 inhibitor SB203580 (10 μM, 1 h) and JNK inhibitor SP600125 (20 μM, 1 h) on p38 and JNK1/2 phosphorylation and α-SMA level after TGF-β1 treatment. C. Impact of Notch3 siRNA knockdown or inhibitor DAPT (10 μM, 1 h) on TGF-β1-induced α-SMA expression. Cells were transfected with a scrambled control (siCtrl) or Notch3 siRNA (siNotch3) for 24 h prior to treatment with TGF-β1 for 48 h. D. Effect of DAPT pretreatment (10 μM, 1 h) on TGF-β1 (1 h)-induced p38 and JNK1/2 activation, as well as ERK1/2 activity. E. Effect of p38 inhibitor SB203580 (10 μM, 1 h), JNK inhibitor SP600125 (20 μM, 1 h) and ERK inhibitor U0126 (10 μM, 1 h) on Notch3 expression after TGF-β1 treatment for 48 h. The protein levels of α-SMA and Notch3 were quantified, normalized by β-actin and presented relative to the one treated with TGF-β1 alone (100%) as means ± SEM (n = 3-4). *, P

    Article Snippet: Western analysis was performed as previously described [ ] by using antibodies against Notch3 (sc-5593), α-SMA and pro-collagen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (Sigma), and p-JNK1/2, p-p38, p-ERK1/2, JNK1/2, p38, ERK1/2, and β-actin (Cell signaling), and a chemiluminescence detection kit (LumiGLO® Reagent and Peroxide, Cell signaling).

    Techniques: Expressing, Transfection, Activation Assay, Activity Assay

    TGF-β1 induces differentiation, p38 and JNK1/2 phosphorylation, and Notch3 expression in IMR-90 fibroblasts Cells were serum-starved overnight prior to treatment with TGF-β1 (200 pM) for the indicated duration. A. Western analysis of α-SMA levels. B. Immunofluorescent analysis of α-SMA expression. Green, α-SMA; blue, nuclei; bar size, 100 μm. C. Western analysis of p38, JNK1/2 and ERK1/2 phosphorylation. D. Western analysis of Notch3 and Notch1 levels. E. RT-PCR analysis of HES1 and HRT1 mRNA levels. The bands were quantified, normalized by β-actin or the respective unphosphorylated protein, and presented relative to 0 h (100%) as means ± SEM (n = 3-4). *, P

    Journal: Oncotarget

    Article Title: Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2

    doi: 10.18632/oncotarget.9667

    Figure Lengend Snippet: TGF-β1 induces differentiation, p38 and JNK1/2 phosphorylation, and Notch3 expression in IMR-90 fibroblasts Cells were serum-starved overnight prior to treatment with TGF-β1 (200 pM) for the indicated duration. A. Western analysis of α-SMA levels. B. Immunofluorescent analysis of α-SMA expression. Green, α-SMA; blue, nuclei; bar size, 100 μm. C. Western analysis of p38, JNK1/2 and ERK1/2 phosphorylation. D. Western analysis of Notch3 and Notch1 levels. E. RT-PCR analysis of HES1 and HRT1 mRNA levels. The bands were quantified, normalized by β-actin or the respective unphosphorylated protein, and presented relative to 0 h (100%) as means ± SEM (n = 3-4). *, P

    Article Snippet: Western analysis was performed as previously described [ ] by using antibodies against Notch3 (sc-5593), α-SMA and pro-collagen (Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (Sigma), and p-JNK1/2, p-p38, p-ERK1/2, JNK1/2, p38, ERK1/2, and β-actin (Cell signaling), and a chemiluminescence detection kit (LumiGLO® Reagent and Peroxide, Cell signaling).

    Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, p-JNK and p-P38, were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.

    Journal: Chinese Medical Journal

    Article Title: Effect of Phosphorylated-Extracellular Regulated Kinase 1/2 Inhibitor on Retina from Light-induced Photoreceptor Degeneration

    doi: 10.4103/0366-6999.246064

    Figure Lengend Snippet: Light exposure increased the expression of p-MAPKs in the retina. MAPKs activation in retina, represented by the levels of p-ERK1/2, p-JNK and p-P38, were detected with immunofluorescence. The green arrows points to the ONL, and the yellow arrows points to the MAPKs enhancement area after light exposure. p-ERK1/2 (a), p-JNK (b) and p-P38 (c) were elevated in the rat model of light-induced retinal degeneration, mainly in the ONL, on days 3 and 5 when compared with their levels in the normal retina. MAPK: Mitogen-activated protein kinase; p-ERK1/2: Phosphorylated-extracellular regulated kinase 1/2; JNK: c-Jun N-terminal kinase; ONL: Outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer.

    Article Snippet: Antibodies directed against the following proteins were used: p-P38 (1:100, Cat #4511, CST, Boston, MA, USA), p-JNK (1:100, Cat #4668, CST, Boston, MA, USA), or p-ERK1/2 (1:100, Cat #4370, CST, Boston, MA, USA).

    Techniques: Expressing, Activation Assay, Immunofluorescence

    The inhibition of JNK and Nod1 reduced the expression of TNFα and IL-1β in HAPI cells treated with LPS. HAPI cells were pretreated with SP600125 (SP, JNK inhibitor) for 1 h, then treated with LPS. SP600125 treatment reduced the expression of TNFα (A), and IL-1β (B), ** p

    Journal: Biochemical and biophysical research communications

    Article Title: TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

    doi: 10.1016/j.bbrc.2012.03.068

    Figure Lengend Snippet: The inhibition of JNK and Nod1 reduced the expression of TNFα and IL-1β in HAPI cells treated with LPS. HAPI cells were pretreated with SP600125 (SP, JNK inhibitor) for 1 h, then treated with LPS. SP600125 treatment reduced the expression of TNFα (A), and IL-1β (B), ** p

    Article Snippet: The membrane was then blocked and then incubated with primary antibodies overnight at 4°C, including rabbit anti-pNF-κB (1:2000, Cell Signaling, Danvers, MA), mouse anti-NF-κB(1:2000, Millipore, Billerica, MA), mouse anti-pJNK (1:2000, Cell Signaling), rabbit anti-JNK (1:2000, Cell Signaling), rabbit anti-TNFα (1:1000, Millipore, Billerica, MA), rabbit anti-IL-1β (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-TLR-4 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-actin (1:10000, Santa Cruz Biotechnology).

    Techniques: Inhibition, Expressing

    TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, JNK1/2 and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p

    Journal: International Journal of Molecular Sciences

    Article Title: Tubeimoside I Protects Dopaminergic Neurons Against Inflammation-Mediated Damage in Lipopolysaccharide (LPS)-Evoked Model of Parkinson’s Disease in Rats

    doi: 10.3390/ijms19082242

    Figure Lengend Snippet: TBMS1 inhibits the phosphorylation of p38 and ERK1/2 in LPS-exposed BV-2 cells. The BV-2 cells were pretreated for 1 h with TBMS1 (1, 2 and 4 μM), then incubated with LPS (1 μg/mL) for 1 h. ( A ) The protein levels of p38, ERK1/2, JNK1/2 and their phosphorylated forms were tested by western blotting. The phosphorylation of p38 ( B ), ERK1/2 ( C ) and JNK1/2 ( D ) was analyzed relative to β-actin. The results are shown as mean ± SD of three independent experiments. # p

    Article Snippet: After blocking with 5% skim milk, the polyvinylidene difluoride membranes were incubated with primary antibodies against TH (1:1000) (Abcam, Cambridge, UK), OX-42 (1:1000) (Abcam), COX-2 (1:2000) (Abcam), iNOS (1:2000) (Abcam), phospho-AKT (1:2000) (Cell Signaling Technology, MA, USA), phospho-JNK1/2 (1:2000) (Cell Signaling Technology), phospho-ERK1/2 (1:2000) (Cell Signaling Technology), phospho-p38 (1:2,000), phospho-NF-κB p65 (1:2000), JNK1/2 (1:2000), ERK1/2 (1:22,000) (Cell Signaling Technology), p38 (1:2000) (Cell Signaling Technology), AKT (1:2000) (Cell Signaling Technology), NF-κB p65 (1:2000) (Cell Signaling Technology) and β-actin (1:10,000) (Santa Cruz, CA, USA).

    Techniques: Incubation, Western Blot

    Effect of muscle-specific JNK1 deficiency on hepatic lipogenic gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice was measured by quantitative RT-PCR analysis of mRNA. Data for the expression of transcription factors ( C/ebp α, C/ebp β, Ppar γ, and Srebp1 ), coactivators ( Pgc1 α and Pgc1 β), fatty acid synthase ( Fas ), and microsomal triglyceride transfer protein ( Mttp ) mRNA are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Effect of muscle-specific JNK1 deficiency on hepatic lipogenic gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice was measured by quantitative RT-PCR analysis of mRNA. Data for the expression of transcription factors ( C/ebp α, C/ebp β, Ppar γ, and Srebp1 ), coactivators ( Pgc1 α and Pgc1 β), fatty acid synthase ( Fas ), and microsomal triglyceride transfer protein ( Mttp ) mRNA are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    Muscle-specific JNK1 deficiency causes increased diet-induced hepatic steatosis. (A) Chow-fed (ND) and HFD-fed (HF) M WT and M KO mice were fasted overnight. Representative sections of the liver stained with hematoxylin and eosin are presented. Scale bar, 100 μm. (B) The amount of hepatic triglyceride was measured in mice fasted overnight (means ± SD) ( n = 10). Statistically significant differences between M KO and M WT are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Muscle-specific JNK1 deficiency causes increased diet-induced hepatic steatosis. (A) Chow-fed (ND) and HFD-fed (HF) M WT and M KO mice were fasted overnight. Representative sections of the liver stained with hematoxylin and eosin are presented. Scale bar, 100 μm. (B) The amount of hepatic triglyceride was measured in mice fasted overnight (means ± SD) ( n = 10). Statistically significant differences between M KO and M WT are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Mouse Assay, Staining

    Effect of muscle-specific JNK1 deficiency on diet-induced obesity. (A) ITTs of M WT and M KO male mice fed either a chow diet (ND) or an HFD for 16 weeks were performed by intraperitoneal injection of insulin (1.5 U/kg body mass). The concentration of blood glucose was measured (means ± SD) ( n = 10). Statistically significant differences between M KO and M WT are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Effect of muscle-specific JNK1 deficiency on diet-induced obesity. (A) ITTs of M WT and M KO male mice fed either a chow diet (ND) or an HFD for 16 weeks were performed by intraperitoneal injection of insulin (1.5 U/kg body mass). The concentration of blood glucose was measured (means ± SD) ( n = 10). Statistically significant differences between M KO and M WT are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Mouse Assay, Injection, Concentration Assay

    Effect of muscle-specific JNK1 deficiency on blood lipids. (A) HFD-fed M WT and M KO mice were fasted overnight. The amounts of cholesterol, triglyceride, and free fatty acid (FFA) in the blood were measured (means ± SD) ( n = 10). Statistically significant differences between M KO and M WT are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Effect of muscle-specific JNK1 deficiency on blood lipids. (A) HFD-fed M WT and M KO mice were fasted overnight. The amounts of cholesterol, triglyceride, and free fatty acid (FFA) in the blood were measured (means ± SD) ( n = 10). Statistically significant differences between M KO and M WT are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Mouse Assay

    Muscle-specific JNK1 deficiency causes increased diet-induced inflammation of adipose tissue. (A) Chow-fed (ND) and HFD-fed (HF) M WT and M KO mice were fasted overnight. Representative histological sections of epididymal adipose tissue stained with hematoxylin and eosin (left panels) and with an antibody (F4/80) to a macrophage marker (right panels) are presented. Scale bar, 100 μm. (B) Gene expression in epididymal adipose tissue was measured by quantitative RT-PCR analysis of mRNA. The relative mRNA expression level was calculated by normalization of the data to the amount of Gapdh mRNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Muscle-specific JNK1 deficiency causes increased diet-induced inflammation of adipose tissue. (A) Chow-fed (ND) and HFD-fed (HF) M WT and M KO mice were fasted overnight. Representative histological sections of epididymal adipose tissue stained with hematoxylin and eosin (left panels) and with an antibody (F4/80) to a macrophage marker (right panels) are presented. Scale bar, 100 μm. (B) Gene expression in epididymal adipose tissue was measured by quantitative RT-PCR analysis of mRNA. The relative mRNA expression level was calculated by normalization of the data to the amount of Gapdh mRNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Mouse Assay, Staining, Marker, Expressing, Quantitative RT-PCR

    Creation of mice with muscle-specific JNK1 deficiency. (A) Extracts prepared from epididymal fat (white adipose tissue), liver, and muscle (gastrocnemius, quadriceps, and soleus) of Mck-Cre + Jnk1 +/+ (M WT ) mice and Mck-Cre + Jnk1 LoxP/LoxP (M KO ) mice were examined by immunoblot analysis by probing with antibodies to JNK1 and GAPDH. (B) M WT and M KO mice were fed a chow diet (ND) or HFD (HF) for 16 weeks and then fasted overnight. JNK protein activity in epididymal fat (adipose tissue), liver, and muscle was measured in a protein kinase (KA) assay using c-Jun and [γ- 32 P]ATP as substrates. The cell extracts used for the protein kinase assay were also examined by immunoblot analysis by probing with antibodies to JNK1 and GAPDH. (C) Male M WT and M KO mice (8 to 10 weeks old) were fed either a chow diet (ND) or HFD for 16 weeks. The body weight of the mice was measured (means ± standard deviations [SD]) ( n = 10). No significant difference between the body weights of M WT and M KO mice was detected ( P > 0.05). (D) Chow-fed M WT and M KO mice were fasted overnight and then treated by intraperitoneal injection of 1.5 mU/kg insulin. Extracts prepared from gastrocnemius muscle at 10 min postinjection were examined by immunoblot analysis using antibodies to the insulin receptor (IR), IRS-1, phospho-Tyr, and IRS-1-Ser 307 . IP, immunoprecipitation.

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Creation of mice with muscle-specific JNK1 deficiency. (A) Extracts prepared from epididymal fat (white adipose tissue), liver, and muscle (gastrocnemius, quadriceps, and soleus) of Mck-Cre + Jnk1 +/+ (M WT ) mice and Mck-Cre + Jnk1 LoxP/LoxP (M KO ) mice were examined by immunoblot analysis by probing with antibodies to JNK1 and GAPDH. (B) M WT and M KO mice were fed a chow diet (ND) or HFD (HF) for 16 weeks and then fasted overnight. JNK protein activity in epididymal fat (adipose tissue), liver, and muscle was measured in a protein kinase (KA) assay using c-Jun and [γ- 32 P]ATP as substrates. The cell extracts used for the protein kinase assay were also examined by immunoblot analysis by probing with antibodies to JNK1 and GAPDH. (C) Male M WT and M KO mice (8 to 10 weeks old) were fed either a chow diet (ND) or HFD for 16 weeks. The body weight of the mice was measured (means ± standard deviations [SD]) ( n = 10). No significant difference between the body weights of M WT and M KO mice was detected ( P > 0.05). (D) Chow-fed M WT and M KO mice were fasted overnight and then treated by intraperitoneal injection of 1.5 mU/kg insulin. Extracts prepared from gastrocnemius muscle at 10 min postinjection were examined by immunoblot analysis using antibodies to the insulin receptor (IR), IRS-1, phospho-Tyr, and IRS-1-Ser 307 . IP, immunoprecipitation.

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Mouse Assay, Activity Assay, Protein Kinase Assay, Injection, Immunoprecipitation

    Effect of muscle-specific deficiency of JNK1 on insulin sensitivity. (A to F) Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp with conscious chow-fed (ND) and HFD-fed (HF) M KO and M WT mice. (A) Insulin-stimulated whole-body glucose turnover. (B) Whole-body glycolysis. (C) Whole-body glycogen plus lipid synthesis. (D) Basal HGP. (E) Insulin-stimulated rate of HGP during the clamp assay. (F) Hepatic insulin action expressed as the insulin-mediated percent suppression of basal HGP. The data presented are the means ± standard errors for approximately six to nine experiments. Statistically significant differences between M KO mice and M WT mice are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Effect of muscle-specific deficiency of JNK1 on insulin sensitivity. (A to F) Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp with conscious chow-fed (ND) and HFD-fed (HF) M KO and M WT mice. (A) Insulin-stimulated whole-body glucose turnover. (B) Whole-body glycolysis. (C) Whole-body glycogen plus lipid synthesis. (D) Basal HGP. (E) Insulin-stimulated rate of HGP during the clamp assay. (F) Hepatic insulin action expressed as the insulin-mediated percent suppression of basal HGP. The data presented are the means ± standard errors for approximately six to nine experiments. Statistically significant differences between M KO mice and M WT mice are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Mouse Assay

    Effect of muscle-specific JNK1 deficiency on insulin-stimulated AKT activation. Chow-fed (ND) and HFD-fed (HF) M WT and M KO mice were fasted overnight and treated by intraperitoneal injection of insulin (1.5 U/kg body mass). Extracts prepared from gastrocnemius muscle (A), liver (B), and epididymal adipose tissue (C) at 10 min postinjection were examined by immunoblot analysis with antibodies to JNK1, AKT, phospho-AKT, and GAPDH.

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Effect of muscle-specific JNK1 deficiency on insulin-stimulated AKT activation. Chow-fed (ND) and HFD-fed (HF) M WT and M KO mice were fasted overnight and treated by intraperitoneal injection of insulin (1.5 U/kg body mass). Extracts prepared from gastrocnemius muscle (A), liver (B), and epididymal adipose tissue (C) at 10 min postinjection were examined by immunoblot analysis with antibodies to JNK1, AKT, phospho-AKT, and GAPDH.

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Activation Assay, Mouse Assay, Injection

    Effect of muscle-specific JNK1 deficiency on hepatic inflammatory gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice fasted overnight was measured by quantitative RT-PCR analysis of mRNA (TaqMan assays). Data for the expression of Tnf α, Il6 , Ifn γ, Cd68 , Icam1 , Lyzs , and cytochrome p450 2E1 ( Cye2e1 ) mRNAs are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance ▿

    doi: 10.1128/MCB.01162-09

    Figure Lengend Snippet: Effect of muscle-specific JNK1 deficiency on hepatic inflammatory gene expression. Gene expression in the liver of chow-fed (ND) and HFD-fed (HF) M WT and M KO mice fasted overnight was measured by quantitative RT-PCR analysis of mRNA (TaqMan assays). Data for the expression of Tnf α, Il6 , Ifn γ, Cd68 , Icam1 , Lyzs , and cytochrome p450 2E1 ( Cye2e1 ) mRNAs are presented. The relative mRNA expression level was calculated by normalization of the data to the amount of 18S RNA in each sample (means ± SD) ( n = 6 to ∼8). Statistically significant differences are indicated (*, P

    Article Snippet: Extracts (20 to 50 μg of protein) were examined by protein immunoblot analysis by probing with antibodies to AKT, phospho-Thr308 AKT, and phospho-Ser473 AKT (Cell Signaling); IRS1 ( ); phospho-Ser307 IRS1 (Millipore); and insulin receptor β subunit, JNK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    ERK1/2 and ERK5 are implicated in the regulation of c-Jun expression in response to EGF. Wild-type (wt), jnk − / − , and erk5 − / − MEFs without treatment (−) or pretreated (+) with PD184352 (2 μM) or SP600125

    Journal: Molecular and Cellular Biology

    Article Title: Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

    doi: 10.1128/MCB.25.9.3784-3792.2005

    Figure Lengend Snippet: ERK1/2 and ERK5 are implicated in the regulation of c-Jun expression in response to EGF. Wild-type (wt), jnk − / − , and erk5 − / − MEFs without treatment (−) or pretreated (+) with PD184352 (2 μM) or SP600125

    Article Snippet: The membranes were incubated with 5% nonfat dry milk or 3% bovine serum albumin at 4°C overnight and then probed with polyclonal antibodies to c-Jun (Cell Signaling), MAPKAPK2 (Cell Signaling), ERK5 , ERK1/2 (Santa Cruz), JNK (Santa Cruz), or p38 MAPK (Santa Cruz).

    Techniques: Expressing

    p38 MAPK contributes to the regulation of c-Jun expression in response to UV. Wild-type (wt) (A and C) and jnk − / − (B and C) MEFs without treatment (−) or pretreated (+) with SB203580 (SB; 10 μM) were UV radiated

    Journal: Molecular and Cellular Biology

    Article Title: Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

    doi: 10.1128/MCB.25.9.3784-3792.2005

    Figure Lengend Snippet: p38 MAPK contributes to the regulation of c-Jun expression in response to UV. Wild-type (wt) (A and C) and jnk − / − (B and C) MEFs without treatment (−) or pretreated (+) with SB203580 (SB; 10 μM) were UV radiated

    Article Snippet: The membranes were incubated with 5% nonfat dry milk or 3% bovine serum albumin at 4°C overnight and then probed with polyclonal antibodies to c-Jun (Cell Signaling), MAPKAPK2 (Cell Signaling), ERK5 , ERK1/2 (Santa Cruz), JNK (Santa Cruz), or p38 MAPK (Santa Cruz).

    Techniques: Expressing

    Model of the regulation of the promoter of the c- jun gene by MAPKs. MAPK activators include MEK1 and MEK2 for ERK1/2, MEK5 for ERK5, MKK4 and MKK7 for JNK, and MKK3 and MKK6 for p38 MAPK. UV activates JNK and p38 MAPK, leading to the subsequent increase

    Journal: Molecular and Cellular Biology

    Article Title: Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

    doi: 10.1128/MCB.25.9.3784-3792.2005

    Figure Lengend Snippet: Model of the regulation of the promoter of the c- jun gene by MAPKs. MAPK activators include MEK1 and MEK2 for ERK1/2, MEK5 for ERK5, MKK4 and MKK7 for JNK, and MKK3 and MKK6 for p38 MAPK. UV activates JNK and p38 MAPK, leading to the subsequent increase

    Article Snippet: The membranes were incubated with 5% nonfat dry milk or 3% bovine serum albumin at 4°C overnight and then probed with polyclonal antibodies to c-Jun (Cell Signaling), MAPKAPK2 (Cell Signaling), ERK5 , ERK1/2 (Santa Cruz), JNK (Santa Cruz), or p38 MAPK (Santa Cruz).

    Techniques:

    p38 MAPK antagonizes the effect of JNK on the transcriptional regulation of c- jun following EGF treatment. Wild-type (wt) (A, B, D, and E) and jnk − / − (C and D) MEFs without treatment (−) or pretreated (+) with SB203580

    Journal: Molecular and Cellular Biology

    Article Title: Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

    doi: 10.1128/MCB.25.9.3784-3792.2005

    Figure Lengend Snippet: p38 MAPK antagonizes the effect of JNK on the transcriptional regulation of c- jun following EGF treatment. Wild-type (wt) (A, B, D, and E) and jnk − / − (C and D) MEFs without treatment (−) or pretreated (+) with SB203580

    Article Snippet: The membranes were incubated with 5% nonfat dry milk or 3% bovine serum albumin at 4°C overnight and then probed with polyclonal antibodies to c-Jun (Cell Signaling), MAPKAPK2 (Cell Signaling), ERK5 , ERK1/2 (Santa Cruz), JNK (Santa Cruz), or p38 MAPK (Santa Cruz).

    Techniques:

    The targeting of c- jun mRNA by MAPK cascades via the TRE and the MEF2 binding sites. jnk − / − (A), erk5 − / − (B), and wild-type (wt) (C) fibroblasts were transiently transfected as described in Materials and Methods. Levels

    Journal: Molecular and Cellular Biology

    Article Title: Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

    doi: 10.1128/MCB.25.9.3784-3792.2005

    Figure Lengend Snippet: The targeting of c- jun mRNA by MAPK cascades via the TRE and the MEF2 binding sites. jnk − / − (A), erk5 − / − (B), and wild-type (wt) (C) fibroblasts were transiently transfected as described in Materials and Methods. Levels

    Article Snippet: The membranes were incubated with 5% nonfat dry milk or 3% bovine serum albumin at 4°C overnight and then probed with polyclonal antibodies to c-Jun (Cell Signaling), MAPKAPK2 (Cell Signaling), ERK5 , ERK1/2 (Santa Cruz), JNK (Santa Cruz), or p38 MAPK (Santa Cruz).

    Techniques: Binding Assay, Transfection

    Rosiglitazone counteracts JNK-induced β-cell dysfunction. GTT ( A ) and the corresponding area under the curve (AUC) for the first 60 min ( B ), glucose-stimulated insulin secretion ( C ), the corresponding AUC for the first 15 min ( D ), and ITT ( E ) performed in control and MKK7D 2-month-old mice treated for 10 days with rosiglitazone (Rosi) or PBS as indicated. F : Insulin secretion of pancreatic islets isolated from control and MKK7D mice treated with PBS or rosiglitazone. Islets were stimulated with glucose and insulin as indicated. G : Immunoblot analysis performed in extracts of pancreatic islets from control and MKK7D mice treated with rosiglitazone and insulin (Ins) (20 min) as indicated. The ratios of phospho-Akt (P-Akt) and phospho-JNK (P-JNK) to AKT and JNK, respectively, are represented in the right panel . Assays were performed with at least 10 animals per group and repeated at least 3 times. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: Rosiglitazone counteracts JNK-induced β-cell dysfunction. GTT ( A ) and the corresponding area under the curve (AUC) for the first 60 min ( B ), glucose-stimulated insulin secretion ( C ), the corresponding AUC for the first 15 min ( D ), and ITT ( E ) performed in control and MKK7D 2-month-old mice treated for 10 days with rosiglitazone (Rosi) or PBS as indicated. F : Insulin secretion of pancreatic islets isolated from control and MKK7D mice treated with PBS or rosiglitazone. Islets were stimulated with glucose and insulin as indicated. G : Immunoblot analysis performed in extracts of pancreatic islets from control and MKK7D mice treated with rosiglitazone and insulin (Ins) (20 min) as indicated. The ratios of phospho-Akt (P-Akt) and phospho-JNK (P-JNK) to AKT and JNK, respectively, are represented in the right panel . Assays were performed with at least 10 animals per group and repeated at least 3 times. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Mouse Assay, Isolation

    Tamoxifen-inducible JNK activation in adult mice causes glucose intolerance. A : GTT of control and MKK7DTam 8-week-old mice before treatment with tamoxifen. B : Immunoblot analysis of GFP, MKK7, phosphorylated JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of tamoxifen-treated control and MKK7DTam mice. C , D , and E : GTT, ITT, and glucose-stimulated insulin secretion, respectively, in tamoxifen-treated control and MKK7DTam mice. F : Insulin secretion of pancreatic islets isolated from tamoxifen-treated control and MKK7DTam mice stimulated with glucose and insulin as indicated. Unless indicated, assays were performed 2 weeks after tamoxifen treatment with 10 animals per group. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: Tamoxifen-inducible JNK activation in adult mice causes glucose intolerance. A : GTT of control and MKK7DTam 8-week-old mice before treatment with tamoxifen. B : Immunoblot analysis of GFP, MKK7, phosphorylated JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of tamoxifen-treated control and MKK7DTam mice. C , D , and E : GTT, ITT, and glucose-stimulated insulin secretion, respectively, in tamoxifen-treated control and MKK7DTam mice. F : Insulin secretion of pancreatic islets isolated from tamoxifen-treated control and MKK7DTam mice stimulated with glucose and insulin as indicated. Unless indicated, assays were performed 2 weeks after tamoxifen treatment with 10 animals per group. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Mouse Assay, Isolation

    JNK activation in pancreatic β-cells affects neither the morphology nor the insulin content of the islets. Pancreatic histological sections of control and MKK7D animals stained with hematoxylin-eosin ( A ) or analyzed by immunohistochemistry with antibodies against insulin and glucagon ( B ). Average β-cell volume to islet volume (Vv) ( C ) and islet area ( D ), insulin content of pancreas ( E ), pancreas weight relative to body weight ( F ), insulin content in isolated islets ( G ) from control and MKK7D mice. H : Immunoblot analysis of caspase 3 in extracts from isolated islets of control and MKK7D mice. Positive control was an extract from neurons treated overnight with staurosporine. I : Caspase 3 activity in extracts from isolated islets untreated or incubated for 20 h with 10 μmol/L staurosporine (STS) of control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals or 200 islets per group. ** P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells affects neither the morphology nor the insulin content of the islets. Pancreatic histological sections of control and MKK7D animals stained with hematoxylin-eosin ( A ) or analyzed by immunohistochemistry with antibodies against insulin and glucagon ( B ). Average β-cell volume to islet volume (Vv) ( C ) and islet area ( D ), insulin content of pancreas ( E ), pancreas weight relative to body weight ( F ), insulin content in isolated islets ( G ) from control and MKK7D mice. H : Immunoblot analysis of caspase 3 in extracts from isolated islets of control and MKK7D mice. Positive control was an extract from neurons treated overnight with staurosporine. I : Caspase 3 activity in extracts from isolated islets untreated or incubated for 20 h with 10 μmol/L staurosporine (STS) of control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals or 200 islets per group. ** P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Staining, Immunohistochemistry, Isolation, Mouse Assay, Positive Control, Activity Assay, Incubation

    JNK activation in pancreatic β-cells leads to glucose intolerance as a result of an impaired capacity to increase insulinemia in response to hyperglycemia. A : Diagram of the GFP loxP -MKK7D transgene. Transgenic mice constitutively express GFP under the control of the CMV enhancer/chicken β-actin promoter (control mice). When crossed with RIP-Cre mice, the GFP cassette is floxed in pancreatic β-cells, leading to the constitutive expression of MKK7D in this cell type (MKK7D mice). E-GFP-N1 (enhanced green fluorescent protein) encodes a red-shifted variant of wild-type GFP. B : Immunoblot analysis of GFP, MKK7, phospho-JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of control and MKK7D mice (first to fourth panel). Fifth panel shows JNK activity assessed by immunocomplex assay. C : Body weight comparison of control and MKK7D mice. Plasma glucose ( D ) and insulin ( E ) level in 6-h-fasted control and MKK7D mice. GTT ( F ), ITT ( G ), and glucose-stimulated insulin secretion ( H ) in control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals per group. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells leads to glucose intolerance as a result of an impaired capacity to increase insulinemia in response to hyperglycemia. A : Diagram of the GFP loxP -MKK7D transgene. Transgenic mice constitutively express GFP under the control of the CMV enhancer/chicken β-actin promoter (control mice). When crossed with RIP-Cre mice, the GFP cassette is floxed in pancreatic β-cells, leading to the constitutive expression of MKK7D in this cell type (MKK7D mice). E-GFP-N1 (enhanced green fluorescent protein) encodes a red-shifted variant of wild-type GFP. B : Immunoblot analysis of GFP, MKK7, phospho-JNK (P-JNK), and JNK in extracts from isolated pancreatic islets of control and MKK7D mice (first to fourth panel). Fifth panel shows JNK activity assessed by immunocomplex assay. C : Body weight comparison of control and MKK7D mice. Plasma glucose ( D ) and insulin ( E ) level in 6-h-fasted control and MKK7D mice. GTT ( F ), ITT ( G ), and glucose-stimulated insulin secretion ( H ) in control and MKK7D mice. Assays were performed in 3-month-old mice with at least 10 animals per group. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Transgenic Assay, Mouse Assay, Expressing, Variant Assay, Isolation, Activity Assay

    JNK activation in pancreatic β-cells impairs glucose-stimulated insulin secretion. Insulin secretion of pancreatic islets isolated from control and MKK7D mice stimulated with the indicated concentrations of glucose ( A ) and in the presence or absence of JNKi-1 (JNKi) as indicated ( B ). Analysis of GLUT2 expression by immunoblot in extracts from pancreatic islets ( C ) and subcellular localization by immunohistochemistry in pancreatic sections ( D ) of control and MKK7D mice. E : Insulin secretion of islets isolated from control and MKK7D mice in response to 2.8 mmol/L (−) and 24 mmol/L glucose (Glu), succinate, KIC, tolbutamide (Tolb), KCl, and IBMX, as indicated. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells impairs glucose-stimulated insulin secretion. Insulin secretion of pancreatic islets isolated from control and MKK7D mice stimulated with the indicated concentrations of glucose ( A ) and in the presence or absence of JNKi-1 (JNKi) as indicated ( B ). Analysis of GLUT2 expression by immunoblot in extracts from pancreatic islets ( C ) and subcellular localization by immunohistochemistry in pancreatic sections ( D ) of control and MKK7D mice. E : Insulin secretion of islets isolated from control and MKK7D mice in response to 2.8 mmol/L (−) and 24 mmol/L glucose (Glu), succinate, KIC, tolbutamide (Tolb), KCl, and IBMX, as indicated. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Isolation, Mouse Assay, Expressing, Immunohistochemistry

    JNK activation in pancreatic β-cells impairs insulin signaling. A : Insulin-induced insulin secretion of pancreatic islets isolated from control and MKK7D mice in the presence or absence of JNKi-1 (JNKi) as indicated. Secretions were performed in basal glucose concentration (2.8 mmol/L). B : Immunoblot analysis of phospho-Akt (P-Akt) and Akt in extracts of islets from control and MKK7D mice incubated with or without insulin (Ins), as indicated, for 20 min. The ratio of phospho-Akt to total Akt is represented in the bottom panel . C : Quantitative RT-PCR analysis of the indicated insulin target genes in islets from control and MKK7 mice incubated with or without insulin, as indicated, for 3 h. D : Immunoblot analysis of Pdx1 in extracts from pancreatic islets of control and MKK7D mice. The ratio of Pdx1 to loading control is represented in the bottom panel . E : Immunohistochemistry analysis of Pdx1 and insulin in pancreatic sections from control and MKK7D mice untreated (−) or treated with glucose (Glu) or insulin (Ins) for 30 min. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Journal: Diabetes

    Article Title: In Vivo JNK Activation in Pancreatic ?-Cells Leads to Glucose Intolerance Caused by Insulin Resistance in Pancreas

    doi: 10.2337/db12-1097

    Figure Lengend Snippet: JNK activation in pancreatic β-cells impairs insulin signaling. A : Insulin-induced insulin secretion of pancreatic islets isolated from control and MKK7D mice in the presence or absence of JNKi-1 (JNKi) as indicated. Secretions were performed in basal glucose concentration (2.8 mmol/L). B : Immunoblot analysis of phospho-Akt (P-Akt) and Akt in extracts of islets from control and MKK7D mice incubated with or without insulin (Ins), as indicated, for 20 min. The ratio of phospho-Akt to total Akt is represented in the bottom panel . C : Quantitative RT-PCR analysis of the indicated insulin target genes in islets from control and MKK7 mice incubated with or without insulin, as indicated, for 3 h. D : Immunoblot analysis of Pdx1 in extracts from pancreatic islets of control and MKK7D mice. The ratio of Pdx1 to loading control is represented in the bottom panel . E : Immunohistochemistry analysis of Pdx1 and insulin in pancreatic sections from control and MKK7D mice untreated (−) or treated with glucose (Glu) or insulin (Ins) for 30 min. Assays were performed in 3-month-old mice and repeated at least 3 times. * P

    Article Snippet: Dual specificity mitogen-activated protein kinase kinase 7 (MKK7), JNK, green fluorescent protein (GFP), Akt, phospho-Akt, caspase 3, phospho-JNK, Pdx1, and GLUT2 were detected using the antibodies sc-7104, sc-474 (Santa Cruz Biotechnology), 11814460001 (Roche), 9272S, 4060P, 9662 (Cell Signaling), 36-9300 (Invitrogen), 07-696 (Upstate), and AB1342 (Chemicon), respectively.

    Techniques: Activation Assay, Isolation, Mouse Assay, Concentration Assay, Incubation, Quantitative RT-PCR, Immunohistochemistry

    Schematic illustration of possible anti-hyperalgesia mechanisms of WTD under neuropathic pain conditions Peripheral nerve injury induces pro-inflammatory cytokine such as IL-1β, causing the activation of spinal astrocytic IL-1R/TRAF6/JNK signaling and the subsequent releasing of chemokines such as CCL2 and CXCL1, which further facilitate synaptic transmission and enhance NP [ 19 ]. The effective inhibition IL-1β and its downstream signaling of IL-1R1/TRAF6/JNK, especially IL-1R1 in astrocytes may be involved in the anti-hyperalgesia effect of WTD under NP disorders.

    Journal: Oncotarget

    Article Title: Wu-tou decoction attenuates neuropathic pain via suppressing spinal astrocytic IL-1R1/TRAF6/JNK signaling

    doi: 10.18632/oncotarget.21638

    Figure Lengend Snippet: Schematic illustration of possible anti-hyperalgesia mechanisms of WTD under neuropathic pain conditions Peripheral nerve injury induces pro-inflammatory cytokine such as IL-1β, causing the activation of spinal astrocytic IL-1R/TRAF6/JNK signaling and the subsequent releasing of chemokines such as CCL2 and CXCL1, which further facilitate synaptic transmission and enhance NP [ 19 ]. The effective inhibition IL-1β and its downstream signaling of IL-1R1/TRAF6/JNK, especially IL-1R1 in astrocytes may be involved in the anti-hyperalgesia effect of WTD under NP disorders.

    Article Snippet: When testing, the sections were rinsed in PBS (PH=7.2-7.4) 3 times (15 minutes each), blocked with 8% goat serum or BSA in 0.5% Triton-X-100 (life sciences) for 2 hours at room temperature, then the prepared sections were incubated in block solution for 12 hours at 4°C with primary antibodies of IL-1R1 (1:100, anti-rabbit, Abcam), TRAF6 (1:50, anti-rabbit, Santa Cruz), p-JNK (1:200, anti-rabbit, Santa Cruz), GFAP antibody (1:200, anti-mouse, Cell Signaling Technology) and CD11b (1:75, anti-rat, Abcam).

    Techniques: Activation Assay, Transmission Assay, Inhibition

    Anti-hyperalgesia and chemokines inhibitory effects of WTD co-administrated with specific inhibitors of IL-1R1/TRAF6/JNK signaling Pretreatment of IL1-Ra (100 mg/kg, i.p., 2 hours before), an inhibitor of IL-1R1, with WTD (12.60 g/kg, p.o., 1 hour before) significantly attenuated SNL induced mechanical allodynia, heat hyperalgesia and increased WTD analgesic effect (a, b) . Otherwise, neither co-administration of LV-TRAF6 shRNA (4 μl, intraspinal injection, 3 days prior to test), the specific inhibitor of TRAF6, nor D-JNKI-1 (0.3 mg/kg, i.p., 30 minutes before), the specific inhibitor of JNK with WTD (12.60 g/kg, p.o., 1 hour before) increased WTD's anti-allodynia effect (c, d) . Accordingly, co-administration of IL-Ra, but not LV-TRAF6 shRNA or D-JNKI-1 further reduced CCL2 (e) or CXCL1 (f) expressions. Data are represented as mean ± SEM. (n=6). ### P

    Journal: Oncotarget

    Article Title: Wu-tou decoction attenuates neuropathic pain via suppressing spinal astrocytic IL-1R1/TRAF6/JNK signaling

    doi: 10.18632/oncotarget.21638

    Figure Lengend Snippet: Anti-hyperalgesia and chemokines inhibitory effects of WTD co-administrated with specific inhibitors of IL-1R1/TRAF6/JNK signaling Pretreatment of IL1-Ra (100 mg/kg, i.p., 2 hours before), an inhibitor of IL-1R1, with WTD (12.60 g/kg, p.o., 1 hour before) significantly attenuated SNL induced mechanical allodynia, heat hyperalgesia and increased WTD analgesic effect (a, b) . Otherwise, neither co-administration of LV-TRAF6 shRNA (4 μl, intraspinal injection, 3 days prior to test), the specific inhibitor of TRAF6, nor D-JNKI-1 (0.3 mg/kg, i.p., 30 minutes before), the specific inhibitor of JNK with WTD (12.60 g/kg, p.o., 1 hour before) increased WTD's anti-allodynia effect (c, d) . Accordingly, co-administration of IL-Ra, but not LV-TRAF6 shRNA or D-JNKI-1 further reduced CCL2 (e) or CXCL1 (f) expressions. Data are represented as mean ± SEM. (n=6). ### P

    Article Snippet: When testing, the sections were rinsed in PBS (PH=7.2-7.4) 3 times (15 minutes each), blocked with 8% goat serum or BSA in 0.5% Triton-X-100 (life sciences) for 2 hours at room temperature, then the prepared sections were incubated in block solution for 12 hours at 4°C with primary antibodies of IL-1R1 (1:100, anti-rabbit, Abcam), TRAF6 (1:50, anti-rabbit, Santa Cruz), p-JNK (1:200, anti-rabbit, Santa Cruz), GFAP antibody (1:200, anti-mouse, Cell Signaling Technology) and CD11b (1:75, anti-rat, Abcam).

    Techniques: shRNA, Injection

    WTD decreased IL-1R1, TRAF6 expressions and p-JNK level in the dorsal horn of L5 spinal cord tissue Time course profile of IL-1R1, TRAF6 expressions, and p-JNK levels in the dorsal horn of the L5 spinal cord in sham- or SNL-operated mice (a, c, e) . Chronic administration of WTD (3.15-12.60 g/kg) dose-dependently reduced IL-1R1, TRAF6 expressions and p-JNK levels in spinal cord tissue, while pregabalin (25 mg/kg, p.o.) had no obvious effects on those factors (b, d, f) . Data are represented as mean ± SEM. (n=3). ## P

    Journal: Oncotarget

    Article Title: Wu-tou decoction attenuates neuropathic pain via suppressing spinal astrocytic IL-1R1/TRAF6/JNK signaling

    doi: 10.18632/oncotarget.21638

    Figure Lengend Snippet: WTD decreased IL-1R1, TRAF6 expressions and p-JNK level in the dorsal horn of L5 spinal cord tissue Time course profile of IL-1R1, TRAF6 expressions, and p-JNK levels in the dorsal horn of the L5 spinal cord in sham- or SNL-operated mice (a, c, e) . Chronic administration of WTD (3.15-12.60 g/kg) dose-dependently reduced IL-1R1, TRAF6 expressions and p-JNK levels in spinal cord tissue, while pregabalin (25 mg/kg, p.o.) had no obvious effects on those factors (b, d, f) . Data are represented as mean ± SEM. (n=3). ## P

    Article Snippet: When testing, the sections were rinsed in PBS (PH=7.2-7.4) 3 times (15 minutes each), blocked with 8% goat serum or BSA in 0.5% Triton-X-100 (life sciences) for 2 hours at room temperature, then the prepared sections were incubated in block solution for 12 hours at 4°C with primary antibodies of IL-1R1 (1:100, anti-rabbit, Abcam), TRAF6 (1:50, anti-rabbit, Santa Cruz), p-JNK (1:200, anti-rabbit, Santa Cruz), GFAP antibody (1:200, anti-mouse, Cell Signaling Technology) and CD11b (1:75, anti-rat, Abcam).

    Techniques: Mouse Assay

    WTD decreased spinal astrocytic levels of p-JNK 10 days after surgical operation, p-JNK were primarily localized in astrocyte (a) . Immunofluorescence staining showed WTD (3.15-12.60 g/kg), but not pregabalin (25 mg/kg, p.o.), dose-dependently decreased p-JNK/GFAP-IR cells in the superficial lamina of SNL mice (b) . Data are represented as mean ± SEM. ## P

    Journal: Oncotarget

    Article Title: Wu-tou decoction attenuates neuropathic pain via suppressing spinal astrocytic IL-1R1/TRAF6/JNK signaling

    doi: 10.18632/oncotarget.21638

    Figure Lengend Snippet: WTD decreased spinal astrocytic levels of p-JNK 10 days after surgical operation, p-JNK were primarily localized in astrocyte (a) . Immunofluorescence staining showed WTD (3.15-12.60 g/kg), but not pregabalin (25 mg/kg, p.o.), dose-dependently decreased p-JNK/GFAP-IR cells in the superficial lamina of SNL mice (b) . Data are represented as mean ± SEM. ## P

    Article Snippet: When testing, the sections were rinsed in PBS (PH=7.2-7.4) 3 times (15 minutes each), blocked with 8% goat serum or BSA in 0.5% Triton-X-100 (life sciences) for 2 hours at room temperature, then the prepared sections were incubated in block solution for 12 hours at 4°C with primary antibodies of IL-1R1 (1:100, anti-rabbit, Abcam), TRAF6 (1:50, anti-rabbit, Santa Cruz), p-JNK (1:200, anti-rabbit, Santa Cruz), GFAP antibody (1:200, anti-mouse, Cell Signaling Technology) and CD11b (1:75, anti-rat, Abcam).

    Techniques: Immunofluorescence, Staining, Mouse Assay

    PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Irradiation, Expressing, Plasmid Preparation, Transfection, Positive Control, shRNA, Real-time Polymerase Chain Reaction, Incubation

    Model for the Regulation of c-SRC-Induced JNK Activation following UV Signaling by PP2A Complexes doi:10.1371/journal.pgen.0030218.g009

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: Model for the Regulation of c-SRC-Induced JNK Activation following UV Signaling by PP2A Complexes doi:10.1371/journal.pgen.0030218.g009

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Activation Assay

    Inhibition of JNK Activity by PR55γ Is Dependent upon Ser12 of c-SRC (A) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12A . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK), JNK1 and JNK2 (α-JNK), or FLAG (α-FLAG). (B) GFP-PR55γ vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12D . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), FLAG (α-FLAG), or GFP (α-GFP). (C) U2-OS cells coexpressing PR55γ shRNAs and FLAG-SRC or FLAG- SRC S12A as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. (D) U2-OS cells expressing PR55γ and FLAG-SRC or FLAG- SRC S12D as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. doi:10.1371/journal.pgen.0030218.g008

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: Inhibition of JNK Activity by PR55γ Is Dependent upon Ser12 of c-SRC (A) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12A . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK), JNK1 and JNK2 (α-JNK), or FLAG (α-FLAG). (B) GFP-PR55γ vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12D . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), FLAG (α-FLAG), or GFP (α-GFP). (C) U2-OS cells coexpressing PR55γ shRNAs and FLAG-SRC or FLAG- SRC S12A as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. (D) U2-OS cells expressing PR55γ and FLAG-SRC or FLAG- SRC S12D as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. doi:10.1371/journal.pgen.0030218.g008

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Inhibition, Activity Assay, Plasmid Preparation, Irradiation, Expressing

    PR55γ Regulates JNK Upstream of MKK4 and at the Level or Upstream of c-SRC (A) pJNK and pMKK4 in relation to the total level of unphosphorylated protein in UV irradiated U2-OS cells followed over time (0–60 min) in the presence or absence of PR55γ KD2 vector. (B) U2-OS cells expressing PR55γ KD2 or a control vector were treated with UV and incubated for 60 min. Whole cell extracts were probed with the indicated antibodies. (C) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole-cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells were cotransfected with PR55γ KD2 vector or control vector as indicated and incubated with PP2 for 2 h and UV for 1 h. Whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK) or JNK1 and JNK2 (α-JNK). (E) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of pSuper-c-SRC. Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), or SRC(α- SRC). (F) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of CDC42 V12 (dominant active). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 (α-JNK). (G) Cells were transfected with either the PP2A pool targeting PR55δ vector or control vector and cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). doi:10.1371/journal.pgen.0030218.g004

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Regulates JNK Upstream of MKK4 and at the Level or Upstream of c-SRC (A) pJNK and pMKK4 in relation to the total level of unphosphorylated protein in UV irradiated U2-OS cells followed over time (0–60 min) in the presence or absence of PR55γ KD2 vector. (B) U2-OS cells expressing PR55γ KD2 or a control vector were treated with UV and incubated for 60 min. Whole cell extracts were probed with the indicated antibodies. (C) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole-cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells were cotransfected with PR55γ KD2 vector or control vector as indicated and incubated with PP2 for 2 h and UV for 1 h. Whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK) or JNK1 and JNK2 (α-JNK). (E) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of pSuper-c-SRC. Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), or SRC(α- SRC). (F) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of CDC42 V12 (dominant active). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 (α-JNK). (G) Cells were transfected with either the PP2A pool targeting PR55δ vector or control vector and cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). doi:10.1371/journal.pgen.0030218.g004

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Irradiation, Plasmid Preparation, Expressing, Incubation, Dominant Negative Mutation, Transfection

    PP2A Family Screen (A) Schematic of the PP2A holoenzyme and outline of the B regulatory subunit families. (B) U2-OS cells were transfected with the indicated pSuper constructs and where available cotransfected with an HA-tagged version of the corresponding PP2A B subunit. Immunoblot panels show the efficiency of knockdown in six different pools as judged by the ability to knockdown cotransfected or endogenous protein (GFP is a transfection control). (C) U2-OS cells were cotransfected with pooled PP2A shRNAs or a control vector. Levels of phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK) were shown in cell lysates for the different samples 60 min after UV treatment of the cells. doi:10.1371/journal.pgen.0030218.g001

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PP2A Family Screen (A) Schematic of the PP2A holoenzyme and outline of the B regulatory subunit families. (B) U2-OS cells were transfected with the indicated pSuper constructs and where available cotransfected with an HA-tagged version of the corresponding PP2A B subunit. Immunoblot panels show the efficiency of knockdown in six different pools as judged by the ability to knockdown cotransfected or endogenous protein (GFP is a transfection control). (C) U2-OS cells were cotransfected with pooled PP2A shRNAs or a control vector. Levels of phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK) were shown in cell lysates for the different samples 60 min after UV treatment of the cells. doi:10.1371/journal.pgen.0030218.g001

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Transfection, Construct, Plasmid Preparation

    PR55γ Regulates c-SRC-Induced JNK Activation (A) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pSUPER-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (B) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pcDNA-HA-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (C) U2-OS cells were transfected with FLAG-SRC or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (D) U2-OS cells were cotransfected with FLAG-SRC, pSUPER-PR55γ, or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (E) U2-OS cells were cotransfected with FLAG-SRC, pcDNA-HA-PR55γ, or a empty vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (F) U2-OS cells expressing pcDNA-HA-hairpins targeting PR55γ or a control vector, and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. (G) U2-OS cells expressing pcDNA-HA-PR55γ or a control vector and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. doi:10.1371/journal.pgen.0030218.g006

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Regulates c-SRC-Induced JNK Activation (A) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pSUPER-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (B) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pcDNA-HA-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (C) U2-OS cells were transfected with FLAG-SRC or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (D) U2-OS cells were cotransfected with FLAG-SRC, pSUPER-PR55γ, or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (E) U2-OS cells were cotransfected with FLAG-SRC, pcDNA-HA-PR55γ, or a empty vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (F) U2-OS cells expressing pcDNA-HA-hairpins targeting PR55γ or a control vector, and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. (G) U2-OS cells expressing pcDNA-HA-PR55γ or a control vector and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. doi:10.1371/journal.pgen.0030218.g006

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Activation Assay, Expressing, Luciferase, Construct, Transfection, Plasmid Preparation, Irradiation, Immunoprecipitation

    EGCG pretreatment of promyelocytic HL-60 inhibits S1P response in macrophage-differentiated HL-60 cells. Notes: Three different conditions were used to evaluate the EGCG impact on the S1P response. Predifferentiation conditions: serum-starved HL-60 cells were treated for 24 hours with various concentrations of EGCG (0–10 μM), then treated for 18 hours with 3 μM PMA, to induce HL-60 cell differentiation and adhesion ( A – C ). Codifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with either 3 μM PMA alone or with a combination of 3 μM PMA and various EGCG concentrations ( D – F ). Postdifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA, then adherent cells were treated for 24 hours with various EGCG concentrations ( G – I ). In all three conditions, adherent cells were then treated with 1 μM S1P for 30 seconds, then cell lysates were isolated, electrophoresed via SDS-PAGE, and immunodetection performed. Scanning densitometry of the autoradiograms was performed, and ratios of P-ERK/ERK, P-JNK/JNK, and P-P38/P38 were obtained. The data shown is representative of three independent experiments. Abbreviations: EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HL, human leukemia; P-P38, phosphorylated P38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; PMA, phorbol-12-myristate-13-acetate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; S1P, sphingosine-1-phosphate.

    Journal: OncoTargets and therapy

    Article Title: Transcriptional targeting of sphingosine-1-phosphate receptor S1P2 by epigallocatechin-3-gallate prevents sphingosine-1-phosphate-mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells

    doi: 10.2147/OTT.S62717

    Figure Lengend Snippet: EGCG pretreatment of promyelocytic HL-60 inhibits S1P response in macrophage-differentiated HL-60 cells. Notes: Three different conditions were used to evaluate the EGCG impact on the S1P response. Predifferentiation conditions: serum-starved HL-60 cells were treated for 24 hours with various concentrations of EGCG (0–10 μM), then treated for 18 hours with 3 μM PMA, to induce HL-60 cell differentiation and adhesion ( A – C ). Codifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with either 3 μM PMA alone or with a combination of 3 μM PMA and various EGCG concentrations ( D – F ). Postdifferentiation conditions: serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA, then adherent cells were treated for 24 hours with various EGCG concentrations ( G – I ). In all three conditions, adherent cells were then treated with 1 μM S1P for 30 seconds, then cell lysates were isolated, electrophoresed via SDS-PAGE, and immunodetection performed. Scanning densitometry of the autoradiograms was performed, and ratios of P-ERK/ERK, P-JNK/JNK, and P-P38/P38 were obtained. The data shown is representative of three independent experiments. Abbreviations: EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HL, human leukemia; P-P38, phosphorylated P38; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; PMA, phorbol-12-myristate-13-acetate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; S1P, sphingosine-1-phosphate.

    Article Snippet: The polyclonal antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (P)-ERK, c-Jun N-terminal kinase (JNK), P-JNK, P38, and P-P38 were all purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Cell Differentiation, Isolation, SDS Page, Immunodetection, Polyacrylamide Gel Electrophoresis

    S1P triggers rapid intracellular signaling through multiple pathways in macrophage-differentiated HL-60 cells. Notes: Serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA to induce HL-60 cell differentiation and adhesion. Adherent cells were then treated with 1 μM S1P for up to 1.5 minutes, following which cell lysates were isolated, electrophoresed via SDS-PAGE, and immunodetected for ( A ) P-ERK, ERK, ( C ) P-JNK, JNK, ( E ) P-P38, and P38 proteins. Quantification was performed by scanning densitometry of the autoradiograms ( B , D , and F ). Abbreviations: ERK, extracellular signal-regulated kinase; HL, human leukemia; JNK, c-Jun N-terminal kinase; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; P-P38, phosphorylated P38; PMA, phorbol-12-myristate-13-acetate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; S1P, sphingosine-1-phosphate; sec, seconds.

    Journal: OncoTargets and therapy

    Article Title: Transcriptional targeting of sphingosine-1-phosphate receptor S1P2 by epigallocatechin-3-gallate prevents sphingosine-1-phosphate-mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells

    doi: 10.2147/OTT.S62717

    Figure Lengend Snippet: S1P triggers rapid intracellular signaling through multiple pathways in macrophage-differentiated HL-60 cells. Notes: Serum-starved HL-60 cells were treated for 18 hours with 3 μM PMA to induce HL-60 cell differentiation and adhesion. Adherent cells were then treated with 1 μM S1P for up to 1.5 minutes, following which cell lysates were isolated, electrophoresed via SDS-PAGE, and immunodetected for ( A ) P-ERK, ERK, ( C ) P-JNK, JNK, ( E ) P-P38, and P38 proteins. Quantification was performed by scanning densitometry of the autoradiograms ( B , D , and F ). Abbreviations: ERK, extracellular signal-regulated kinase; HL, human leukemia; JNK, c-Jun N-terminal kinase; P-ERK, phosphorylated ERK; P-JNK, phosphorylated JNK; P-P38, phosphorylated P38; PMA, phorbol-12-myristate-13-acetate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; S1P, sphingosine-1-phosphate; sec, seconds.

    Article Snippet: The polyclonal antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (P)-ERK, c-Jun N-terminal kinase (JNK), P-JNK, P38, and P-P38 were all purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Cell Differentiation, Isolation, SDS Page, Polyacrylamide Gel Electrophoresis, Size-exclusion Chromatography

    Schematic representation of pretreatment, cotreatment, or postdifferentiation cell treatments with EGCG, on S1P-mediated signaling pathways. Notes: ( A ) Undifferentiated HL60 cells express the putative EGCG receptor 67LR as well as all five S1P receptors. S1P triggers phosphorylation of ERK, P38 MAPK, and JNK. When cells were pretreated with EGCG, S1P-mediated signaling was abrogated and S1P2 gene expression inhibited. ( B ) Differentiation into adherent macrophages was induced with PMA, and only S1P1 , S1P2 , and S1P5 gene expression was increased. When EGCG was added simultaneously with PMA-induced cell differentiation, S1P2 gene expression was inhibited, and only S1P-mediated P38 MAPK phosphorylation was abrogated. ( C ) Once terminal differentiation was achieved, EGCG was ineffective at inhibiting S1P receptor expression or S1P-mediated signaling, partly because the 67LR was no longer expressed. Abbreviations: 67LR, 67kDa laminin receptor; EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; P38 MAPK, P38 mitogen-activated protein kinase; PMA, phorbol-12-myristate-13-acetate; S1P, sphingosine-1-phosphate.

    Journal: OncoTargets and therapy

    Article Title: Transcriptional targeting of sphingosine-1-phosphate receptor S1P2 by epigallocatechin-3-gallate prevents sphingosine-1-phosphate-mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells

    doi: 10.2147/OTT.S62717

    Figure Lengend Snippet: Schematic representation of pretreatment, cotreatment, or postdifferentiation cell treatments with EGCG, on S1P-mediated signaling pathways. Notes: ( A ) Undifferentiated HL60 cells express the putative EGCG receptor 67LR as well as all five S1P receptors. S1P triggers phosphorylation of ERK, P38 MAPK, and JNK. When cells were pretreated with EGCG, S1P-mediated signaling was abrogated and S1P2 gene expression inhibited. ( B ) Differentiation into adherent macrophages was induced with PMA, and only S1P1 , S1P2 , and S1P5 gene expression was increased. When EGCG was added simultaneously with PMA-induced cell differentiation, S1P2 gene expression was inhibited, and only S1P-mediated P38 MAPK phosphorylation was abrogated. ( C ) Once terminal differentiation was achieved, EGCG was ineffective at inhibiting S1P receptor expression or S1P-mediated signaling, partly because the 67LR was no longer expressed. Abbreviations: 67LR, 67kDa laminin receptor; EGCG, epigallocatechin-3-gallate; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; P38 MAPK, P38 mitogen-activated protein kinase; PMA, phorbol-12-myristate-13-acetate; S1P, sphingosine-1-phosphate.

    Article Snippet: The polyclonal antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (P)-ERK, c-Jun N-terminal kinase (JNK), P-JNK, P38, and P-P38 were all purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Expressing, Cell Differentiation