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    Cell Signaling Technology Inc extracellular regulated kinase 1 2 erk1 2
    Phosphorylation of Exo70 by <t>ERK1/2</t> promotes VSV-G exocytosis
    Extracellular Regulated Kinase 1 2 Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 1483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho extracellular regulated kinase 1 2 erk1 2
    METH-induced autophagy involves Akt/mTOR and the ERK pathways in endothelial cells. ( a ) Cells exposed to METH for 3, 6, 12, 24 and 48 h showed an increase in <t>ERK1/2</t> phosphorylation after 6 h of treatment. ( b ) Densitometric analysis of p-ERK1/2 level. Values were normalized against α -tubulin and presented as a ratio compared with the basal level (0 h of treatment). Data represent the means of three independent blots. * P
    Phospho Extracellular Regulated Kinase 1 2 Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 79/100, based on 1222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho extracellular regulated kinase 1 2
    METH-induced autophagy involves Akt/mTOR and the ERK pathways in endothelial cells. ( a ) Cells exposed to METH for 3, 6, 12, 24 and 48 h showed an increase in <t>ERK1/2</t> phosphorylation after 6 h of treatment. ( b ) Densitometric analysis of p-ERK1/2 level. Values were normalized against α -tubulin and presented as a ratio compared with the basal level (0 h of treatment). Data represent the means of three independent blots. * P
    Anti Phospho Extracellular Regulated Kinase 1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc extracellular regulated kinase 1 2 inhibitor u0126
    Effect of Forskolin on activation of downstream signaling pathways CREB, CEBPβ, and <t>ERK1/2</t> in M0 macrophages (Control), or during M2 (IL-4) vs. M1 (IFNγ) polarizing conditions. Bone marrow-derived macrophages were grown for 5 days in the presence of M-CSF and were treated with Forskolin, or IL4, or IFNγ, or Forskolin/IL4, or Forskolin/IFNγ, or Forskolin/IL4/IFNγ for 24 h as in Figure 1 . ERK1/2 inhibitor <t>U0126</t> was used to block ERK1 and ERK2 phosphorylation (see Materials and Methods ). Expressions of CREB, CEBPβ, and ERK1/2 and their phosphorylated forms (p-CREB, p-CEBPβ, and p-ERK1/2) were analyzed by Western blot as described in Section “ Materials and Methods .” β-Actin was used as a loading control. (A,C,E) Quantitative analysis of relative expression levels of CREB, CEBPβ, and ERK normalized to β-actin is shown. Representative image is shown in Figure S1 in Supplementary Material. (B,D,F) Quantitative analysis of relative expression levels of p-CREB, p-CEBPβ, and p-ERK normalized to total CREB, CEBPβ, and ERK1/2, respectively, is shown. Representative image is shown in Figure S1 in Supplementary Material. In panels (A–D) , mean ± SE of three separate experiments is shown. Abbreviations: IFN, IFNγ; IL4, IL-4; F, Forskolin; U, U0126.
    Extracellular Regulated Kinase 1 2 Inhibitor U0126, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc t202 y204 phosphorylated extracellular regulated kinase 1 2
    Effect of Forskolin on activation of downstream signaling pathways CREB, CEBPβ, and <t>ERK1/2</t> in M0 macrophages (Control), or during M2 (IL-4) vs. M1 (IFNγ) polarizing conditions. Bone marrow-derived macrophages were grown for 5 days in the presence of M-CSF and were treated with Forskolin, or IL4, or IFNγ, or Forskolin/IL4, or Forskolin/IFNγ, or Forskolin/IL4/IFNγ for 24 h as in Figure 1 . ERK1/2 inhibitor <t>U0126</t> was used to block ERK1 and ERK2 phosphorylation (see Materials and Methods ). Expressions of CREB, CEBPβ, and ERK1/2 and their phosphorylated forms (p-CREB, p-CEBPβ, and p-ERK1/2) were analyzed by Western blot as described in Section “ Materials and Methods .” β-Actin was used as a loading control. (A,C,E) Quantitative analysis of relative expression levels of CREB, CEBPβ, and ERK normalized to β-actin is shown. Representative image is shown in Figure S1 in Supplementary Material. (B,D,F) Quantitative analysis of relative expression levels of p-CREB, p-CEBPβ, and p-ERK normalized to total CREB, CEBPβ, and ERK1/2, respectively, is shown. Representative image is shown in Figure S1 in Supplementary Material. In panels (A–D) , mean ± SE of three separate experiments is shown. Abbreviations: IFN, IFNγ; IL4, IL-4; F, Forskolin; U, U0126.
    T202 Y204 Phosphorylated Extracellular Regulated Kinase 1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies against extracellular regulated kinase 1 2 erk
    Effect of Forskolin on activation of downstream signaling pathways CREB, CEBPβ, and <t>ERK1/2</t> in M0 macrophages (Control), or during M2 (IL-4) vs. M1 (IFNγ) polarizing conditions. Bone marrow-derived macrophages were grown for 5 days in the presence of M-CSF and were treated with Forskolin, or IL4, or IFNγ, or Forskolin/IL4, or Forskolin/IFNγ, or Forskolin/IL4/IFNγ for 24 h as in Figure 1 . ERK1/2 inhibitor <t>U0126</t> was used to block ERK1 and ERK2 phosphorylation (see Materials and Methods ). Expressions of CREB, CEBPβ, and ERK1/2 and their phosphorylated forms (p-CREB, p-CEBPβ, and p-ERK1/2) were analyzed by Western blot as described in Section “ Materials and Methods .” β-Actin was used as a loading control. (A,C,E) Quantitative analysis of relative expression levels of CREB, CEBPβ, and ERK normalized to β-actin is shown. Representative image is shown in Figure S1 in Supplementary Material. (B,D,F) Quantitative analysis of relative expression levels of p-CREB, p-CEBPβ, and p-ERK normalized to total CREB, CEBPβ, and ERK1/2, respectively, is shown. Representative image is shown in Figure S1 in Supplementary Material. In panels (A–D) , mean ± SE of three separate experiments is shown. Abbreviations: IFN, IFNγ; IL4, IL-4; F, Forskolin; U, U0126.
    Antibodies Against Extracellular Regulated Kinase 1 2 Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc pospho erk1 2
    Effect of Forskolin on activation of downstream signaling pathways CREB, CEBPβ, and <t>ERK1/2</t> in M0 macrophages (Control), or during M2 (IL-4) vs. M1 (IFNγ) polarizing conditions. Bone marrow-derived macrophages were grown for 5 days in the presence of M-CSF and were treated with Forskolin, or IL4, or IFNγ, or Forskolin/IL4, or Forskolin/IFNγ, or Forskolin/IL4/IFNγ for 24 h as in Figure 1 . ERK1/2 inhibitor <t>U0126</t> was used to block ERK1 and ERK2 phosphorylation (see Materials and Methods ). Expressions of CREB, CEBPβ, and ERK1/2 and their phosphorylated forms (p-CREB, p-CEBPβ, and p-ERK1/2) were analyzed by Western blot as described in Section “ Materials and Methods .” β-Actin was used as a loading control. (A,C,E) Quantitative analysis of relative expression levels of CREB, CEBPβ, and ERK normalized to β-actin is shown. Representative image is shown in Figure S1 in Supplementary Material. (B,D,F) Quantitative analysis of relative expression levels of p-CREB, p-CEBPβ, and p-ERK normalized to total CREB, CEBPβ, and ERK1/2, respectively, is shown. Representative image is shown in Figure S1 in Supplementary Material. In panels (A–D) , mean ± SE of three separate experiments is shown. Abbreviations: IFN, IFNγ; IL4, IL-4; F, Forskolin; U, U0126.
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    Cell Signaling Technology Inc phosphorylated erk1 2
    Levels of phosphoextracellular signal-regulated kinase <t>(ERK1/2</t> p44/p42) in mouse 3T3-L1 adipocytes. Cells were pretreated for 24 h with alliin 0.1 mM and subsequently exposed to 100 ng/mL of lipopolysaccharides (LPS) for 1 h afterward. (a) Representative Western blot with phospho-ERK1/2 and ERK1/2 antibodies; (b) protein levels of phospho-ERK1/2 and ERK1/2 in total cell extracts. CT control; AU arbitrary units. Data are expressed as mean ± standard deviations (SD) of three independent experiments * P ≤ 0.05; ** P ≤ 0.01.
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    Cell Signaling Technology Inc phosho erk1 2
    Levels of phosphoextracellular signal-regulated kinase <t>(ERK1/2</t> p44/p42) in mouse 3T3-L1 adipocytes. Cells were pretreated for 24 h with alliin 0.1 mM and subsequently exposed to 100 ng/mL of lipopolysaccharides (LPS) for 1 h afterward. (a) Representative Western blot with phospho-ERK1/2 and ERK1/2 antibodies; (b) protein levels of phospho-ERK1/2 and ERK1/2 in total cell extracts. CT control; AU arbitrary units. Data are expressed as mean ± standard deviations (SD) of three independent experiments * P ≤ 0.05; ** P ≤ 0.01.
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    Cell Signaling Technology Inc phosphor erk1 2
    The involvement of p38/GSK3β/Nrf-2 axis for cytoprotective effects of PGC-1α. To understand the molecular mechanism for cytoprotective effects of PGC-1α, the activation of Keap1-, GSK3β- or Hrd1 ( A ) and MAPKs ( B ), as an upstream signal molecules of Nrf-2, were compared to Mock and PGC-1α cells for indicated time points after H 2 O 2 treatment. To further elucidate the specificity of p38-mediated signal cascade for cytoprotective effects of PGC-1α cells, p38 specific effects were analyzed by western blotting ( C ) and MTT assay ( D ) in PGC-1α cells treated with H 2 O 2 in the presence or absence of p38 inhibitor (SB203580) for 2 h or 4 h, respectively. Inhibitor of <t>ERK1/2</t> (PD98059) was used as non-effective control on PGC-1α effect. Activation of p38 or inactivation of GSK3β was checked with phosphor specific antibody at Thr180/Tyr182 residue or at Ser9 residue, respectively. Full-length blots of each tested protein are reported in Supplementary Figure S7 . Error bars denote the mean ± S.D. of triplicate samples. MTT assay was performed to n = 7. * p
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    Cell Signaling Technology Inc antiphospho erk1 2
    ( A ) Phenobarbital and phenytoin decrease mRNA levels for BDNF and NT-3 and decrease phosphorylation of c-RAF, <t>ERK1/2,</t> and AKT in the neonatal brain. P7 pups received i.p. injection of phenobarbital (50 mg/kg), phenytoin (40 mg/kg), or vehicle (Co). Brain tissue from the thalamus was dissected at the various times indicated. Decreased density of the BDNF- and NT-3-specific bands is evident at 6, 12, and 24 h after administration of AEDs. Immunoblotting was performed with <t>antiphospho-RAF,</t> antiphospho-ERK1/2, antiphospho-AKT, ERK1/2 (phosphorylation state independent), or AKT (phosphorylation state independent) antibodies. There is a decrease in the levels of p-RAF, p-ERK1/2, and p-AKT at 6 h after injection of AEDs, whereas ERK1/2 and AKT (phosphorylation independent) are unaffected. ( B ) Quantitation of suppression by AEDs of mRNA levels for BDNF and NT-3 in the thalamus of infant rats. P7 pups received injection of phenobarbital (50 mg/kg, n = 9), phenytoin (50 mg/kg, n = 9), valproate (200 mg/kg, n = 9), or vehicle ( n = 9) and were killed at 6, 12, or 24 h after treatment ( n = 3 per group). mRNA levels for BDNF, NT-3, and β-actin were analyzed by means of PAGE and densitometrically quantitated. Values represent mean normalized ratios of the BDNF and NT-3 bands to β-actin ( n = 3 per point ± SEM). ANOVA revealed that there was a significant effect of treatment with AEDs on BDNF [ F (1,12) valproate = 29.79, P
    Antiphospho Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc erk1 2 137f5
    ( A ) Phenobarbital and phenytoin decrease mRNA levels for BDNF and NT-3 and decrease phosphorylation of c-RAF, <t>ERK1/2,</t> and AKT in the neonatal brain. P7 pups received i.p. injection of phenobarbital (50 mg/kg), phenytoin (40 mg/kg), or vehicle (Co). Brain tissue from the thalamus was dissected at the various times indicated. Decreased density of the BDNF- and NT-3-specific bands is evident at 6, 12, and 24 h after administration of AEDs. Immunoblotting was performed with <t>antiphospho-RAF,</t> antiphospho-ERK1/2, antiphospho-AKT, ERK1/2 (phosphorylation state independent), or AKT (phosphorylation state independent) antibodies. There is a decrease in the levels of p-RAF, p-ERK1/2, and p-AKT at 6 h after injection of AEDs, whereas ERK1/2 and AKT (phosphorylation independent) are unaffected. ( B ) Quantitation of suppression by AEDs of mRNA levels for BDNF and NT-3 in the thalamus of infant rats. P7 pups received injection of phenobarbital (50 mg/kg, n = 9), phenytoin (50 mg/kg, n = 9), valproate (200 mg/kg, n = 9), or vehicle ( n = 9) and were killed at 6, 12, or 24 h after treatment ( n = 3 per group). mRNA levels for BDNF, NT-3, and β-actin were analyzed by means of PAGE and densitometrically quantitated. Values represent mean normalized ratios of the BDNF and NT-3 bands to β-actin ( n = 3 per point ± SEM). ANOVA revealed that there was a significant effect of treatment with AEDs on BDNF [ F (1,12) valproate = 29.79, P
    Erk1 2 137f5, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc erk1 2 sirnas
    ERK 1/2 is implicated in 5FU toxicity on endothelial cells. (A) Representative images and percentage of EA.hy926 cells stained for SA β‐gal after the following treatments: no treatment (CTR); exposure to 5FU; pre‐incubation with GLP‐1 followed by exposure to 5FU; transfection with <t>ERK1/2</t> <t>siRNA</t> (siERK), pre‐incubation with GLP‐1 and then exposure to 5FU; transfection with sham siRNA (sham); transfection with siERK. Magnification of pictures is 200×, and bars correspond to 50 μm. (B, C) Representative western blots for ERK1/2 and eNOS (B) and optical density (OD) of the protein bands normalized to that of actin (C) after the treatments described in (A). Graphs show mean ± SEM of five independent experiments. Comparisons were made by means of ANOVA followed by post hoc Tukey's multiple comparisons test. * Statistically significant versus CTR, # statistically significant versus 5FU and ¤ statistically significant versus siERK + GLP‐1 + FU.
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    Cell Signaling Technology Inc erk1 2 inhibitor
    <t>ERK1/2</t> activation was enhanced by S1P in LPS-challenged cells. The cells were incubated with inhibitory reagent against ERK1/2 (U0126) prior to LPS and S1P challenge. The induction of IL-6 (A) and IL-8 (B) was determined in culture supernatants by Luminex technology. The level of phosphorylated ERK1/2 was determined by FACE (Active Motif), as described in the Material and methods section, in cells following LPS or S1P/LPS challenge (C). Data are presented as means ± SD of triplicate determinations. Statistically significant ( p
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    Cell Signaling Technology Inc phospholyration erk1 2
    <t>ERK1/2</t> activation was enhanced by S1P in LPS-challenged cells. The cells were incubated with inhibitory reagent against ERK1/2 (U0126) prior to LPS and S1P challenge. The induction of IL-6 (A) and IL-8 (B) was determined in culture supernatants by Luminex technology. The level of phosphorylated ERK1/2 was determined by FACE (Active Motif), as described in the Material and methods section, in cells following LPS or S1P/LPS challenge (C). Data are presented as means ± SD of triplicate determinations. Statistically significant ( p
    Phospholyration Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc erk1 2 u0126
    <t>ERK1/2</t> activation was enhanced by S1P in LPS-challenged cells. The cells were incubated with inhibitory reagent against ERK1/2 (U0126) prior to LPS and S1P challenge. The induction of IL-6 (A) and IL-8 (B) was determined in culture supernatants by Luminex technology. The level of phosphorylated ERK1/2 was determined by FACE (Active Motif), as described in the Material and methods section, in cells following LPS or S1P/LPS challenge (C). Data are presented as means ± SD of triplicate determinations. Statistically significant ( p
    Erk1 2 U0126, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc α erk1 2
    Reduction of Enah downregulates <t>p-Erk1/2,</t> p-AKT, and p-p65 and inhibits EMT (epithelial-mesenchymal transition) progress in GC cells. a Decreased protein levels of p-Erk1/2, p-AKT, p-p65, Vimentin, Fibronectin, and increased protein level of E-cadherin in LV-shEnah cells compared with LV-shcontrol cells. No significant difference was detected in the protein expression of p-STAT3. b Morphology of MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells as visualized using phase-contrast microscopy (magnification × 200). c Immunofluorescence analysis of E-cadherin and Vimentin expression in MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells. * p
    α Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc unphosphorylated erk1 2
    Reduction of Enah downregulates <t>p-Erk1/2,</t> p-AKT, and p-p65 and inhibits EMT (epithelial-mesenchymal transition) progress in GC cells. a Decreased protein levels of p-Erk1/2, p-AKT, p-p65, Vimentin, Fibronectin, and increased protein level of E-cadherin in LV-shEnah cells compared with LV-shcontrol cells. No significant difference was detected in the protein expression of p-STAT3. b Morphology of MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells as visualized using phase-contrast microscopy (magnification × 200). c Immunofluorescence analysis of E-cadherin and Vimentin expression in MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells. * p
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    Cell Signaling Technology Inc dp erk1 2
    MKP3 is dependent on <t>ERK1/2</t> but not Akt activation . (A) Beads soaked with specific inhibitors were placed in the caudal hindbrain or in the presumptive MHB of HH7 + -8 explanted embryos. Pharmacological treatments were as follows: DMSO (a), FGFR inhibitor SU5402 (b), ERK1/2 inhibitor PD184352 (c), PI3K inhibitor LY294002 (d). Explants were incubated during 6 h at 38°C. (B) Jurkat cells (left side lanes) and HH7-8 explanted embryos (right side lanes) were treated with DMSO, SU5402, PD184352 or LY294002 and analyzed by western blot for total and phosphorylated forms of Akt and ERK1/2. PD184352 treatment impeded ERK1/2 phosphorylation without affecting the PI3K-Akt pathway and, conversely, LY294002 treatment abolished Akt phosphorylation without affecting the Ras-ERK1/2 pathway. Embryos are shown in whole-mount with anterior to the top.
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    Cell Signaling Technology Inc phosphorilated erk1 2
    MKP3 is dependent on <t>ERK1/2</t> but not Akt activation . (A) Beads soaked with specific inhibitors were placed in the caudal hindbrain or in the presumptive MHB of HH7 + -8 explanted embryos. Pharmacological treatments were as follows: DMSO (a), FGFR inhibitor SU5402 (b), ERK1/2 inhibitor PD184352 (c), PI3K inhibitor LY294002 (d). Explants were incubated during 6 h at 38°C. (B) Jurkat cells (left side lanes) and HH7-8 explanted embryos (right side lanes) were treated with DMSO, SU5402, PD184352 or LY294002 and analyzed by western blot for total and phosphorylated forms of Akt and ERK1/2. PD184352 treatment impeded ERK1/2 phosphorylation without affecting the PI3K-Akt pathway and, conversely, LY294002 treatment abolished Akt phosphorylation without affecting the Ras-ERK1/2 pathway. Embryos are shown in whole-mount with anterior to the top.
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    Cell Signaling Technology Inc erk1 2 no 9212
    MKP3 is dependent on <t>ERK1/2</t> but not Akt activation . (A) Beads soaked with specific inhibitors were placed in the caudal hindbrain or in the presumptive MHB of HH7 + -8 explanted embryos. Pharmacological treatments were as follows: DMSO (a), FGFR inhibitor SU5402 (b), ERK1/2 inhibitor PD184352 (c), PI3K inhibitor LY294002 (d). Explants were incubated during 6 h at 38°C. (B) Jurkat cells (left side lanes) and HH7-8 explanted embryos (right side lanes) were treated with DMSO, SU5402, PD184352 or LY294002 and analyzed by western blot for total and phosphorylated forms of Akt and ERK1/2. PD184352 treatment impeded ERK1/2 phosphorylation without affecting the PI3K-Akt pathway and, conversely, LY294002 treatment abolished Akt phosphorylation without affecting the Ras-ERK1/2 pathway. Embryos are shown in whole-mount with anterior to the top.
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    Cell Signaling Technology Inc erk1 2 specific sirnas
    MKP3 is dependent on <t>ERK1/2</t> but not Akt activation . (A) Beads soaked with specific inhibitors were placed in the caudal hindbrain or in the presumptive MHB of HH7 + -8 explanted embryos. Pharmacological treatments were as follows: DMSO (a), FGFR inhibitor SU5402 (b), ERK1/2 inhibitor PD184352 (c), PI3K inhibitor LY294002 (d). Explants were incubated during 6 h at 38°C. (B) Jurkat cells (left side lanes) and HH7-8 explanted embryos (right side lanes) were treated with DMSO, SU5402, PD184352 or LY294002 and analyzed by western blot for total and phosphorylated forms of Akt and ERK1/2. PD184352 treatment impeded ERK1/2 phosphorylation without affecting the PI3K-Akt pathway and, conversely, LY294002 treatment abolished Akt phosphorylation without affecting the Ras-ERK1/2 pathway. Embryos are shown in whole-mount with anterior to the top.
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    Effect of depleting GGA1 and GGA2 on α 2B -AR-mediated signaling. ( A ) HEK293 cells were transfected with control shRNA or individual GGA shRNA for 36 h. After starvation for 3 h, the cells were stimulated with UK14304 at the concentration of 1 μM for 5 min at 37 °C. <t>ERK1/2</t> activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel is a representative blot of ERK1/2 activation and lower panel shows total ERK1/2 expression. ( B ) Quantitative data shown in A). The data shown are percentages of the mean value obtained from cells transfected with control shRNA and are presented as the mean ± S.E. of at least three experiments. * p
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    Effect of depleting GGA1 and GGA2 on α 2B -AR-mediated signaling. ( A ) HEK293 cells were transfected with control shRNA or individual GGA shRNA for 36 h. After starvation for 3 h, the cells were stimulated with UK14304 at the concentration of 1 μM for 5 min at 37 °C. <t>ERK1/2</t> activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel is a representative blot of ERK1/2 activation and lower panel shows total ERK1/2 expression. ( B ) Quantitative data shown in A). The data shown are percentages of the mean value obtained from cells transfected with control shRNA and are presented as the mean ± S.E. of at least three experiments. * p
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    Cell Signaling Technology Inc erk1 2 inhibitor pd98059
    The expression of inflammatory cytokines in response to Ang II with or without the presence of MAPK and NF-κB inhibitors. mRNA expression of IL-8, MCP-1 and IL-6 was measured by real-time PCR 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580 ( A ) <t>ERK1/2</t> inhibitor <t>PD98059</t> ( B ) JNK inhibitor SP600125 ( C ) and NF-κB inhibitor BAY11-7082 ( D ) all at a final concentration of 10 μM. ( E ) The protein expression levels of IL-8, MCP-1 and IL-6 were measured by ELISA 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580, ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and NF-κB inhibitor BAY11-7082, all at a final concentration of 10 μM. The data were shown as mean ± SEM (* p
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    Cell Signaling Technology Inc phosphor erk1 2 thr408
    The expression of inflammatory cytokines in response to Ang II with or without the presence of MAPK and NF-κB inhibitors. mRNA expression of IL-8, MCP-1 and IL-6 was measured by real-time PCR 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580 ( A ) <t>ERK1/2</t> inhibitor <t>PD98059</t> ( B ) JNK inhibitor SP600125 ( C ) and NF-κB inhibitor BAY11-7082 ( D ) all at a final concentration of 10 μM. ( E ) The protein expression levels of IL-8, MCP-1 and IL-6 were measured by ELISA 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580, ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and NF-κB inhibitor BAY11-7082, all at a final concentration of 10 μM. The data were shown as mean ± SEM (* p
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    Cell Signaling Technology Inc pharmacologic erk1 2 inhibitor
    Recombinant human (rh) RELM-β has mitogenic effects on human lung microvascular endothelial cells (HMVEC-Ls) and human pulmonary artery smooth muscle cells (HPASMCs) and activates <t>ERK1/2.</t> HMVEC-Ls ( A ) and HPASMCs ( B ) were serum and growth factor
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    Cell Signaling Technology Inc erk1 2 inhibitor uo126
    Ciprofloxacin induces <t>Erk1/2</t> activation and has no effect on TGFβ/Smad signaling. (A) Cells were treated with 50 μ g/ml of ciprofloxacin and P-Erk1/2 levels were analyzed after 12 h; bar graph on the right represents relative quantification of 4 experiments. (B) Cells were pretreated with Erk inhibitor <t>UO126</t> and mRNA levels of MMP1 were analyzed after 48 h of ciprofloxacin treatment (50 μ g/ml) (n=3). * P≤0.05 and ** P≤0.01. (C) Healthy cells and systemic sclerosis dermal fibroblasts were pretreated with 50 μ g/ml of ciprofloxacin overnight then with 2.5 ng/ml of TGFβ and levels of P-Smad3, total Smad2/3, P-Smad1 and total Smad1 were analyzed by western blot analysis. Representative blots from 3 experiments are shown. Cx, ciprofloxacin; NS, healthy cells; SSc, systemic sclerosis; c, control.
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    Cell Signaling Technology Inc erk1 2 phosphorylation
    PLX8394 effectively inhibits <t>ERK1/2</t> signaling in patient tumors comparable to dabrafenib/trametinib treatment A. H E and IHC analysis of pERK1/2 staining from a representative mutant BRAF patient sample (TJU-MEL-27A) treated with either DMSO, vemurafenib (1 μM), combo (1 μM dabrafenib/16 nM trametinib) or PLX8394 (0.5 μM). B. Quantitation of A across a panel of 6 different mutant BRAF melanoma patient samples. C. Western blot analysis of ERK1/2 signaling and PARP cleavage from a representative patient sample (TJU-MEL-27A). D. Western blot quantitation of the normalized pERK1/2 to ERK2 signal from 5 patient samples. E. RPPA data from mutant BRAF patient samples were analyzed via GSEA. Patient explants treated with vemurafenib (left) and PLX8394 (right), were grouped and compared to DMSO treated samples. Enrichment of the Programmed Cell Death/Apoptosis and Cell Cycle Arrest GO pathways and corresponding changes in RPPA determined protein levels compared to DMSO are shown. Pathway nodes and protein levels for all treatments are on the same scale (bottom left).
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    Cell Signaling Technology Inc erk1 2 inhibitor pb98059
    Effect of specific MAPK inhibitors on telomerase. For specific inhibition of p38 activation, 10 µM SB203580 or 20 µM SB202190, of JNK activation, 5 µM SP600125 or 20 µM JNK inhibitor V were used, respectively. 10 µM U0126 or 40 µM <t>PB98059</t> were used for <t>ERK1/2</t> inhibition. Cells were pre-treated with the inhibitors and subsequently exposed to MTBITC or the solvent control (0.1% DMSO). a) Immunoblot analysis shows effective inhibition of the target proteins. β-actin was used as loading control b) Pre-treated cells were analysed for full length hTERT mRNA expression after 1 h exposure to 25 µM MTBITC. PBDG was used as reference gene. mRNA expression was calculated relative to the corresponding solvent control (n = 3). c+d) Pre-treated cells were assayed for telomerase enzyme activity after 3 h (d) or 24 h (e) MTBITC exposure using the TRAP-ELISA assay. Telomerase activity was calculated relative to the corresponding solvent control; bars are mean ± SEM (n = 3). Inhibitors 3×: combination of SB203580, SP600125 and U0126.
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    Cell Signaling Technology Inc phos erk1 2
    Antcin M activates Nrf2-dependent anti-oxidant defense in HNDFs A. To determine the free-radical scavenging effect of ANM, cell-free DPPH assay was performed. NAC and RES were used as positive controls. B. , C. To quantify the mRNA expression levels of HO-1 and NQO-1, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 12 h. Total RNA was extracted and subjected to Q-PCR analysis. Relative mRNA levels were normalized with β-actin mRNA. D. To determine the protein expression levels of HO-1, NQO-1 and Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) for 24 h. Total cell lysates were prepared and subjected to western blot analysis to monitor the expression levels of HO-1, NQO-1 and Nrf2. E. To determine the Nrf2 transcriptional activity, HNDFs were transiently transfected with ARE promoter construct using lipofectamine and incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 6 h. Cell lysates were mixed with luciferase reagents and quantified using an illuminometer. Relative ARE promoter activity was calculated by dividing the relative luciferace unit (RLU) of treated cells by RLU of untreated cells (NG). F. To determine the nuclear localization of Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence or HG (30 mM) for 2 h. The protein expression and localization of Nrf2 was measured by immunofluorescence using Nrf2 specific primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). The subcellular and nuclear localization of Nrf2 was photographed using a fluoroscence microscope. DAPI (1 μM) was used to stain the nucleus. G. HNDFs were pre-incubated with AKT, <t>ERK1/2,</t> JNK/SAPK and p38 MAPK inhbitors LY294002 (LY, 30 μM), PD98059 (PD, 30 μM), SP600125 (SP, 30 μM) and SB203580 (SB, 30 μM), respectively for 2 h and then incubated with ANM (10 μM) in the presence of HG (30 mM) 2 h. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis. GAPDH and histone H3 served as internal controls for the cytoplasmic and nuclear fraction, respectively. H. The Keap-1 protein expression level was determined by western blotting. I. Effect of ANM on ubiquitination of Keap-1. Equivalent amount of proteins were immune-precipitated with Keap-1 antibody and visualized by western blotting with ubiquitin antibody. Histogram shows the percentage of ubiquinated Keap-1. Results expressed as mean ± S.E.M of three indipendent expriments. Statistical significance was set at Ф P
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    Antcin M activates Nrf2-dependent anti-oxidant defense in HNDFs A. To determine the free-radical scavenging effect of ANM, cell-free DPPH assay was performed. NAC and RES were used as positive controls. B. , C. To quantify the mRNA expression levels of HO-1 and NQO-1, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 12 h. Total RNA was extracted and subjected to Q-PCR analysis. Relative mRNA levels were normalized with β-actin mRNA. D. To determine the protein expression levels of HO-1, NQO-1 and Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) for 24 h. Total cell lysates were prepared and subjected to western blot analysis to monitor the expression levels of HO-1, NQO-1 and Nrf2. E. To determine the Nrf2 transcriptional activity, HNDFs were transiently transfected with ARE promoter construct using lipofectamine and incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 6 h. Cell lysates were mixed with luciferase reagents and quantified using an illuminometer. Relative ARE promoter activity was calculated by dividing the relative luciferace unit (RLU) of treated cells by RLU of untreated cells (NG). F. To determine the nuclear localization of Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence or HG (30 mM) for 2 h. The protein expression and localization of Nrf2 was measured by immunofluorescence using Nrf2 specific primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). The subcellular and nuclear localization of Nrf2 was photographed using a fluoroscence microscope. DAPI (1 μM) was used to stain the nucleus. G. HNDFs were pre-incubated with AKT, <t>ERK1/2,</t> JNK/SAPK and p38 MAPK inhbitors LY294002 (LY, 30 μM), PD98059 (PD, 30 μM), SP600125 (SP, 30 μM) and SB203580 (SB, 30 μM), respectively for 2 h and then incubated with ANM (10 μM) in the presence of HG (30 mM) 2 h. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis. GAPDH and histone H3 served as internal controls for the cytoplasmic and nuclear fraction, respectively. H. The Keap-1 protein expression level was determined by western blotting. I. Effect of ANM on ubiquitination of Keap-1. Equivalent amount of proteins were immune-precipitated with Keap-1 antibody and visualized by western blotting with ubiquitin antibody. Histogram shows the percentage of ubiquinated Keap-1. Results expressed as mean ± S.E.M of three indipendent expriments. Statistical significance was set at Ф P
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    Image Search Results


    Phosphorylation of Exo70 by ERK1/2 promotes VSV-G exocytosis

    Journal: Developmental Cell

    Article Title: ERK1/2 Regulate Exocytosis through Direct Phosphorylation of the Exocyst Component Exo70

    doi: 10.1016/j.devcel.2012.03.005

    Figure Lengend Snippet: Phosphorylation of Exo70 by ERK1/2 promotes VSV-G exocytosis

    Article Snippet: The sources of the antibodies used were as follows: monoclonal antibodies against Flag M2 (Sigma-Aldrich), ERK1/2, Phospho-ERK1/2 and ERK1/2 substrate (Cell Signal, Inc.).

    Techniques:

    ERK1/2 phosphorylate Exo70 at Serine 250 in response to EGF

    Journal: Developmental Cell

    Article Title: ERK1/2 Regulate Exocytosis through Direct Phosphorylation of the Exocyst Component Exo70

    doi: 10.1016/j.devcel.2012.03.005

    Figure Lengend Snippet: ERK1/2 phosphorylate Exo70 at Serine 250 in response to EGF

    Article Snippet: The sources of the antibodies used were as follows: monoclonal antibodies against Flag M2 (Sigma-Aldrich), ERK1/2, Phospho-ERK1/2 and ERK1/2 substrate (Cell Signal, Inc.).

    Techniques:

    The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: The HDV replication from genomic RNA to antigenomic RNA is not enhanced by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT (lanes 4 to 7) or pCDSHDAg S177A (lanes 8 to 10) together with 4 μg pCDm2G (lanes 3 and 5 to 10) and combined with an increasing dose (0.4 μg or 2 μg) of HA-AcMEK1 plasmids (lanes 6, 7, 9, and 10). Four days after transfection, RNA and protein lysates were prepared from the transfected cells. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected with DIG-labeled HDV antigenomic and genomic RNA probes in Northern blotting. The lower gel in panel A shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. The RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: Transfection, Labeling, Northern Blot, Staining, Western Blot, Positive Control

    HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: HDV replication from antigenomic RNA to genomic RNA is modulated by ERK1/2-phosphorylated SHDAg at Ser-177. HEK293T cells were transiently transfected with 4 μg pCDSHDAg WT or pCDSHDAg S177A together with 4 μg pCDm2AG and combined with an increasing dose (0.4 μg or 2 μg) of pHA-AcMEK1 plasmid. Four days after transfection, RNA and protein lysates were prepared from the transfected cells. The DIG-labeled HDV antigenomic and genomic RNA transcribed in vitro from plasmids pCD2G and pCD2AG were used as probes for Northern blot analysis. (A and B) The HDV genomic RNA (A) and antigenomic RNA (B) were detected by Northern blotting. The lower gel in panel B shows a longer exposure. (C) Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading. (D) The total lysates were analyzed by Western blotting using antibodies that recognize HA-AcMEK1, pERK1/2, ERK1/2, pSer-177, or SHDAg. “Positive” indicates RNA samples extracted from HDV-replicating cells. RNA was loaded as a positive control to detect genomic or antigenomic RNA in the Northern blotting.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: Transfection, Plasmid Preparation, Labeling, In Vitro, Northern Blot, Staining, Western Blot, Positive Control

    Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: Liquid chromatography-tandem mass spectroscopy analysis shows that the in vivo phosphorylation of SHDAg at Ser-177 is increased with HA-AcMEK1 expression in a dose-dependent manner. (A) HEK293T cells were transiently transfected with 8 μg pFlag-SHDAg WT (FSHDAg WT ) in the presence or absence of different amounts (0.4 μg or 2 μg) of pHA-AcMEK1. After 48 h, the total input lysates were analyzed by 10% SDS-PAGE and probed with HA, ERK1/2, pERK1/2, or Flag antibody. (B) In immunoprecipitation experiments, the eluted Flag-SHDAg was analyzed by 10% SDS-PAGE, and the gel was stained with Coomassie blue. The obvious bands of Flag-SHDAg in the gel were subjected to trypsin in-gel digestion and analyzed using a Q-STARXL Q-TOF mass spectrometer (see Materials and Methods). (C and D) The intensities of the nonphosphorylated ( m/z = 1035.97) (C) and phosphorylated ( m/z = 1075.97) (D) 161 GAPGGGFVPNLQGVPESPFSR 181 peptides were detected using XIC analysis in a Q-STARXL Q-TOF mass spectrometer. Results without MEK1 induction (a) and with induction by 0.4 μg (b) and 2 μg (c) of MEK1 are shown. The intensity of phosphorylated peptides increased with HA-AcMEK1 induction in a dose-dependent manner.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: Liquid Chromatography, Tandem Mass Spectroscopy, In Vivo, Expressing, Transfection, SDS Page, Immunoprecipitation, Staining, Mass Spectrometry

    U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: U0126 treatment inhibits ERK1/2-mediated SHDAgSer-177 phosphorylation and HDV replication without MEK1 overexpression. Huh7 cells were DNA transfected with pCDSHDAg WT and pCDm2AG plasmids. The cells (lanes 3 and 4) were pretreated with 1 μM or 10 μM U0126 for 2 h before transfection, and the treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected with Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: Over Expression, Transfection, Western Blot, Northern Blot, Staining

    ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: ERK1/2 interacts with SHDAg in vivo. pFlag-SHDAg WT (FSHDAg WT ), pFlag-SHDAg 177C , and pFlag-SHDAg S2C were expressed transiently in HEK293T cells for 48 h. The cell fractions were prepared and coimmunoprecipitated (IP) using immobilized anti-Flag antibody-bound resin. Whole-cell extracts before immunoprecipitation were also analyzed (lanes 1 and 3, indicated as input). Input lysates (left panel) and IP lysates (right panel) were resolved by 10% SDS-PAGE and analyzed by Western blotting using antibodies that recognized Flag-SHDAg, CDK1/2, CDK5, p38, or ERK1/2. “Positive” indicates the total cell lysate loading as the control in the Western blotting.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: In Vivo, Immunoprecipitation, SDS Page, Western Blot

    The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: The pS177 antibody specifically recognizes HA-AcMEK1-induced phosphorylation of SHDAg at Ser-177. HEK293T cells were transfected with pFlag-SHDAg WT or pFlag-SHDAg S177A plasmid and cotransfected in the presence or absence of pHA-AcMEK1 plasmid. After 48 h, the cell lysates prepared from HEK293T cells were treated with λ-phosphatase at 4°C for 1 h or left untreated. The lysates were resolved by 10% SDS-PAGE and probed with mouse polyclonal phospho-Ser-177 antibody, Flag, ERK1/2, pERK1/2, or HA antibody.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: Transfection, Plasmid Preparation, SDS Page

    Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: Both Flag-ERK1 and Flag-ERK2 phosphorylate Flag-SHDAg at Ser-177 in the in vitro kinase assay. (A) Full-length Flag-SHDAg WT (lanes 5 to 9) and Flag-SHDAg S177A (lane 10) were used as the substrates in the in vitro kinase reaction mixtures containing the immunoprecipitated Flag-ERK1 (lanes 6, 7, and 10) or Flag-ERK2 (lanes 8, 9, and 10). ATP was not added in the in vitro kinase reaction in lanes 7 and 9. The phosphorylation of SHDAg was probed with pS177 antibody (middle panel), and Flag-ERK1/2 and Flag-SHDAg proteins were visualized with Flag antibody (upper and lower panels) in stained Western blots. (B) Silver staining of Flag-ERK1/2 and Flag-SHDAg confirmed the high purity of kinases and substrates in the kinase assay reactions.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: In Vitro, Kinase Assay, Immunoprecipitation, Staining, Western Blot, Silver Staining

    U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

    Journal: Journal of Virology

    Article Title: ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication ▿

    doi: 10.1128/JVI.00656-08

    Figure Lengend Snippet: U0126 treatment downregulates ERK1/2-mediated phosphorylation of SHDAg at Ser-177 and inhibits HDV replication from antigenomic RNA to genomic RNA. HEK293T cells were DNA transfected with pHA-AcMEK1 and pCDSHDAg WT plasmids combined with RNA transfection. The dimer antigenomic RNA was prepared in the in vitro transcript from the pCDm2AG vector. The cells (lanes 2 and 4) were pretreated with 10 μM U0126 for 2 h before transfection, and treatment was continued for 72 h. (A) Protein samples were prepared, and the Western blot was probed with antibodies that recognize pERK1/2, ERK1/2, pSer-177, or SHDAg. (B) Total RNA was extracted, and HDV genomic or antigenomic RNA was detected using Northern blotting. Ethidium bromide-stained 18S rRNA is shown as a control for RNA loading.

    Article Snippet: Whereas CDK1, CDK5, and p38 were not detected in any of the anti-Flag immunoprecipitates, ERK1/2 was present in the immunoprecipitates from the cells expressing the S177C mutant protein (Fig. , lane 8).

    Techniques: Transfection, In Vitro, Plasmid Preparation, Western Blot, Northern Blot, Staining

    Differential expression patterns of YB-1, c-Kit, MAPT and GST in time course . MCF-7 cells that incubated with 20 nM doxorubicin for the indicated periods of time revealed different kinetic patterns for YB-1 and GST expressions between sensitive and resistant MCF-7 cells. A: Dot blot array analysis on doxorubicin sensitive MCF-7 cells. B: Dot blot array analysis on the doxorubicin resistant MCF-7 cells. A and B: The scale on x-axis is not in proportion with time. In the time course study, actin was employed as a control for normalization, because GAPDH was regulated in doxorubicin resistant MCF-7 cells. C: Effect of doxorubicin on the expression of drug resistance related target proteins YB-1, c-Kit, ERK1/2, ERK3, FAS, MAPT, MDR1, ABCB5 and PARP-1 in the four subtypes of MCF-7 cells. 1, 2 are MCF-7 and MCF-7/vector-YB-1 respectively without treatment of doxorubicin. 3, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (not fused cells); 4, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (fused cells, FACS sorted R2); 5, doxorubicin resistant MCF-7 cell line. Numbers indicate a relative level of protein expression based on the level of intensity of β-actin after normalization.

    Journal: BMC Cancer

    Article Title: p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance

    doi: 10.1186/1471-2407-10-388

    Figure Lengend Snippet: Differential expression patterns of YB-1, c-Kit, MAPT and GST in time course . MCF-7 cells that incubated with 20 nM doxorubicin for the indicated periods of time revealed different kinetic patterns for YB-1 and GST expressions between sensitive and resistant MCF-7 cells. A: Dot blot array analysis on doxorubicin sensitive MCF-7 cells. B: Dot blot array analysis on the doxorubicin resistant MCF-7 cells. A and B: The scale on x-axis is not in proportion with time. In the time course study, actin was employed as a control for normalization, because GAPDH was regulated in doxorubicin resistant MCF-7 cells. C: Effect of doxorubicin on the expression of drug resistance related target proteins YB-1, c-Kit, ERK1/2, ERK3, FAS, MAPT, MDR1, ABCB5 and PARP-1 in the four subtypes of MCF-7 cells. 1, 2 are MCF-7 and MCF-7/vector-YB-1 respectively without treatment of doxorubicin. 3, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (not fused cells); 4, MCF-7/vector-YB-1 with treatment of doxorubicin for 6 h (fused cells, FACS sorted R2); 5, doxorubicin resistant MCF-7 cell line. Numbers indicate a relative level of protein expression based on the level of intensity of β-actin after normalization.

    Article Snippet: The protein extracts were electrophoresed by 12% SDS-PAGE, then electrically transferred onto nitrocellulose filters and probed with the following primary antibodies (specific to the proteins that may associated with acquired drug resistance): YB-1 (dilution factor 1:200, Cell Signalling), c-Kit (1:1000, Cell Signalling); ERK1/2 (1: 1000, Cell Signalling); ERK3 (1: 500, Novus Biologicals); FAS (1: 500, Santa Cruz Biotechnology); MAPT (1: 1500, Proteintech Group); and MDR1 (1: 500, Santa Cruz Biotechnology), ABCB5 (1: 2000, ProSci); PARP-1 (1: 2000, Trevigen); β-Actin (1:5000, Sigma) was used as a loading control.

    Techniques: Expressing, Incubation, Dot Blot, Plasmid Preparation, FACS

    Amylin inhibits osteoclastogenesis ex vivo. (A) In vitro differentiation of osteoclasts is inhibited by amylin at a physiological concentration (10 −10 M). (B) Multinucleated osteoclasts differentiating in the presence of amylin are smaller and have fewer of nuclei. (C) BMMs proliferation and early steps of osteoclast differentiation was not affected by amylin. (•) 10 −10 M amylin; (▴) vehicle. (D) Dentine slice resorption assay in the presence of vehicle or physiological levels of amylin. Resorption is decreased by the presence of amylin in a manner proportional to the decrease in the number of osteoclasts. (E) In vitro differentiation of ERK1/2 dominant negative infected and noninfected osteoclasts in presence of amylin. ERK1/2 is phosphorylated 5 min after stimulation by amylin in noninfected culture, whereas this phosphorylation is abolished in ERK1/2 dominant negative infected cells. (F) Amylin inhibition of osteoclastogenesis is abolished in ERK1/2 dominant negative infected culture. Error bars represent SEM. The asterisks indicate a statistical difference (t ≤ 0.05) between vehicle and treated culture.

    Journal: The Journal of Cell Biology

    Article Title: Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo

    doi: 10.1083/jcb.200312135

    Figure Lengend Snippet: Amylin inhibits osteoclastogenesis ex vivo. (A) In vitro differentiation of osteoclasts is inhibited by amylin at a physiological concentration (10 −10 M). (B) Multinucleated osteoclasts differentiating in the presence of amylin are smaller and have fewer of nuclei. (C) BMMs proliferation and early steps of osteoclast differentiation was not affected by amylin. (•) 10 −10 M amylin; (▴) vehicle. (D) Dentine slice resorption assay in the presence of vehicle or physiological levels of amylin. Resorption is decreased by the presence of amylin in a manner proportional to the decrease in the number of osteoclasts. (E) In vitro differentiation of ERK1/2 dominant negative infected and noninfected osteoclasts in presence of amylin. ERK1/2 is phosphorylated 5 min after stimulation by amylin in noninfected culture, whereas this phosphorylation is abolished in ERK1/2 dominant negative infected cells. (F) Amylin inhibition of osteoclastogenesis is abolished in ERK1/2 dominant negative infected culture. Error bars represent SEM. The asterisks indicate a statistical difference (t ≤ 0.05) between vehicle and treated culture.

    Article Snippet: For Western blot analysis using P-ERK1/2 and ERK1/2 antibodies (Cell Signaling Technology), osteoclasts were lysed in RIPA buffer containing 5 mM iodoacetamide, 10 mM NaF, and 0.4 mM Na2 VO4 with protease inhibitor cocktail (Roche).

    Techniques: Ex Vivo, In Vitro, Concentration Assay, Dominant Negative Mutation, Infection, Inhibition

    Distension of intact ex vivo bladder activates effectors of the mTOR Pathway. A: S6 kinase and ERK1/2 phosphorylation was detected in distended ex vivo bladders by immunofluorescence on cryosections using monoclonal anti-phospho-S6 kinase and -phospho-ERK1/2

    Journal: The American Journal of Pathology

    Article Title: Mammalian Target of Rapamycin (mTOR) Induces Proliferation and De-Differentiation Responses to Three Coordinate Pathophysiologic Stimuli (Mechanical Strain, Hypoxia, and Extracellular Matrix Remodeling) in Rat Bladder Smooth Muscle

    doi: 10.2353/ajpath.2010.080834

    Figure Lengend Snippet: Distension of intact ex vivo bladder activates effectors of the mTOR Pathway. A: S6 kinase and ERK1/2 phosphorylation was detected in distended ex vivo bladders by immunofluorescence on cryosections using monoclonal anti-phospho-S6 kinase and -phospho-ERK1/2

    Article Snippet: Bands were normalized to total actin (Sigma St. Louis, MO), total p70 S6K or pan-ERK1/2 (1:500; Cell Signaling).

    Techniques: Ex Vivo, Immunofluorescence

    Granulocyte colony-stimulating factor (G-CSF) induces the activation of ERK5 in AML cells. (A) The expression levels of ERK5 and ERK1/2 in AML cell lines (Kasumi-1, HL-60, MV4.11) by western blot analysis. (B) The phosphorylation of ERK5 and ERK1/2 in Kasumi-1 cells with the treatment of 10 ng/ml G-CSF.

    Journal: International Journal of Stem Cells

    Article Title: The Pharmacological Inhibition of ERK5 Enhances Apoptosis in Acute Myeloid Leukemia Cells

    doi: 10.15283/ijsc18053

    Figure Lengend Snippet: Granulocyte colony-stimulating factor (G-CSF) induces the activation of ERK5 in AML cells. (A) The expression levels of ERK5 and ERK1/2 in AML cell lines (Kasumi-1, HL-60, MV4.11) by western blot analysis. (B) The phosphorylation of ERK5 and ERK1/2 in Kasumi-1 cells with the treatment of 10 ng/ml G-CSF.

    Article Snippet: The following antibodies were used: ERK5 antibody (Abcam), phospho-ERK1/2 antibody (Cell Signaling Technology), ERK1/2 antibody (Cell Signaling Technology), c-Myc antibody (Abcam). β -tubulin antibody (Abcam) was used to normalize the amount of analyzed samples.

    Techniques: Activation Assay, Expressing, Western Blot

    G-CSF-mediated ERK5 activation induces the proliferation of AML cells. (A, D) XMD8-92 suppressed the G-CSF-stimulated phosphorylation of ERK5 but not affected ERK1/2 in Kasumi-1 (A) and HL-60 (D) cells. (B, E) PD184352, as an inhibitor of ERK1/2, was not inhibited the G-CSF-stimulated activation of ERK5 in Kasumi-1 (B) and HL-60 (E) cells. (C, F) The cell proliferation induced by with or without G-CSF was suppressed by the treatment of XMD8-92 in Kasumi-1 (C) and HL-60 (F) cells, respectively. Data were obtained from three replicate experiments for the cell counting kit-8 assay. Data represent mean±SD. **p

    Journal: International Journal of Stem Cells

    Article Title: The Pharmacological Inhibition of ERK5 Enhances Apoptosis in Acute Myeloid Leukemia Cells

    doi: 10.15283/ijsc18053

    Figure Lengend Snippet: G-CSF-mediated ERK5 activation induces the proliferation of AML cells. (A, D) XMD8-92 suppressed the G-CSF-stimulated phosphorylation of ERK5 but not affected ERK1/2 in Kasumi-1 (A) and HL-60 (D) cells. (B, E) PD184352, as an inhibitor of ERK1/2, was not inhibited the G-CSF-stimulated activation of ERK5 in Kasumi-1 (B) and HL-60 (E) cells. (C, F) The cell proliferation induced by with or without G-CSF was suppressed by the treatment of XMD8-92 in Kasumi-1 (C) and HL-60 (F) cells, respectively. Data were obtained from three replicate experiments for the cell counting kit-8 assay. Data represent mean±SD. **p

    Article Snippet: The following antibodies were used: ERK5 antibody (Abcam), phospho-ERK1/2 antibody (Cell Signaling Technology), ERK1/2 antibody (Cell Signaling Technology), c-Myc antibody (Abcam). β -tubulin antibody (Abcam) was used to normalize the amount of analyzed samples.

    Techniques: Activation Assay, Cell Counting

    METH-induced autophagy involves Akt/mTOR and the ERK pathways in endothelial cells. ( a ) Cells exposed to METH for 3, 6, 12, 24 and 48 h showed an increase in ERK1/2 phosphorylation after 6 h of treatment. ( b ) Densitometric analysis of p-ERK1/2 level. Values were normalized against α -tubulin and presented as a ratio compared with the basal level (0 h of treatment). Data represent the means of three independent blots. * P

    Journal: Cell Death & Disease

    Article Title: Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

    doi: 10.1038/cddis.2014.64

    Figure Lengend Snippet: METH-induced autophagy involves Akt/mTOR and the ERK pathways in endothelial cells. ( a ) Cells exposed to METH for 3, 6, 12, 24 and 48 h showed an increase in ERK1/2 phosphorylation after 6 h of treatment. ( b ) Densitometric analysis of p-ERK1/2 level. Values were normalized against α -tubulin and presented as a ratio compared with the basal level (0 h of treatment). Data represent the means of three independent blots. * P

    Article Snippet: AKT, p-AKT, mTOR, p-mTOR, ERK1/2, p-ERK1/2 and LC3 antibodies were from Cell Signaling (Danvers, MA, USA).

    Techniques:

    Levels of phosphorylated and total ERK1/2 protein within lungs subjected to 5 min of low- or high-stretch ventilation. Consistent with findings from Fig. 3 , levels of phospho-ERK1/2 clearly increased with high-stretch ventilation, whereas levels of total ERK1/2 within the same lungs were unaltered; n = 4. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Kinetic profiling of in vivo lung cellular inflammatory responses to mechanical ventilation

    doi: 10.1152/ajplung.00048.2015

    Figure Lengend Snippet: Levels of phosphorylated and total ERK1/2 protein within lungs subjected to 5 min of low- or high-stretch ventilation. Consistent with findings from Fig. 3 , levels of phospho-ERK1/2 clearly increased with high-stretch ventilation, whereas levels of total ERK1/2 within the same lungs were unaltered; n = 4. * P

    Article Snippet: With the use of a previously published technique ( , ), lung cell suspensions were also stained with AF-647-conjugated antibodies against intracellular phospho-p38 (Thr180/182 ; clone 28B10), phospho-MK2 (Thr334 ; 27B7), phospho-ERK1/2 (Thr202 /Tyr204 ; D13.14.4E), and phospho-NF-κB p65 (Ser536 ; 93H1) (Cell Signaling, Danvers, MA).

    Techniques:

    Effect of Forskolin on activation of downstream signaling pathways CREB, CEBPβ, and ERK1/2 in M0 macrophages (Control), or during M2 (IL-4) vs. M1 (IFNγ) polarizing conditions. Bone marrow-derived macrophages were grown for 5 days in the presence of M-CSF and were treated with Forskolin, or IL4, or IFNγ, or Forskolin/IL4, or Forskolin/IFNγ, or Forskolin/IL4/IFNγ for 24 h as in Figure 1 . ERK1/2 inhibitor U0126 was used to block ERK1 and ERK2 phosphorylation (see Materials and Methods ). Expressions of CREB, CEBPβ, and ERK1/2 and their phosphorylated forms (p-CREB, p-CEBPβ, and p-ERK1/2) were analyzed by Western blot as described in Section “ Materials and Methods .” β-Actin was used as a loading control. (A,C,E) Quantitative analysis of relative expression levels of CREB, CEBPβ, and ERK normalized to β-actin is shown. Representative image is shown in Figure S1 in Supplementary Material. (B,D,F) Quantitative analysis of relative expression levels of p-CREB, p-CEBPβ, and p-ERK normalized to total CREB, CEBPβ, and ERK1/2, respectively, is shown. Representative image is shown in Figure S1 in Supplementary Material. In panels (A–D) , mean ± SE of three separate experiments is shown. Abbreviations: IFN, IFNγ; IL4, IL-4; F, Forskolin; U, U0126.

    Journal: Frontiers in Immunology

    Article Title: Cyclic AMP Pathway Suppress Autoimmune Neuroinflammation by Inhibiting Functions of Encephalitogenic CD4 T Cells and Enhancing M2 Macrophage Polarization at the Site of Inflammation

    doi: 10.3389/fimmu.2018.00050

    Figure Lengend Snippet: Effect of Forskolin on activation of downstream signaling pathways CREB, CEBPβ, and ERK1/2 in M0 macrophages (Control), or during M2 (IL-4) vs. M1 (IFNγ) polarizing conditions. Bone marrow-derived macrophages were grown for 5 days in the presence of M-CSF and were treated with Forskolin, or IL4, or IFNγ, or Forskolin/IL4, or Forskolin/IFNγ, or Forskolin/IL4/IFNγ for 24 h as in Figure 1 . ERK1/2 inhibitor U0126 was used to block ERK1 and ERK2 phosphorylation (see Materials and Methods ). Expressions of CREB, CEBPβ, and ERK1/2 and their phosphorylated forms (p-CREB, p-CEBPβ, and p-ERK1/2) were analyzed by Western blot as described in Section “ Materials and Methods .” β-Actin was used as a loading control. (A,C,E) Quantitative analysis of relative expression levels of CREB, CEBPβ, and ERK normalized to β-actin is shown. Representative image is shown in Figure S1 in Supplementary Material. (B,D,F) Quantitative analysis of relative expression levels of p-CREB, p-CEBPβ, and p-ERK normalized to total CREB, CEBPβ, and ERK1/2, respectively, is shown. Representative image is shown in Figure S1 in Supplementary Material. In panels (A–D) , mean ± SE of three separate experiments is shown. Abbreviations: IFN, IFNγ; IL4, IL-4; F, Forskolin; U, U0126.

    Article Snippet: ERK1/2 inhibitor U0126 was purchased from Cell Signaling, and another ERK1/2 PD184352 inhibitor was purchased from Abcam.

    Techniques: Activation Assay, Derivative Assay, Blocking Assay, Western Blot, Expressing

    Levels of phosphoextracellular signal-regulated kinase (ERK1/2 p44/p42) in mouse 3T3-L1 adipocytes. Cells were pretreated for 24 h with alliin 0.1 mM and subsequently exposed to 100 ng/mL of lipopolysaccharides (LPS) for 1 h afterward. (a) Representative Western blot with phospho-ERK1/2 and ERK1/2 antibodies; (b) protein levels of phospho-ERK1/2 and ERK1/2 in total cell extracts. CT control; AU arbitrary units. Data are expressed as mean ± standard deviations (SD) of three independent experiments * P ≤ 0.05; ** P ≤ 0.01.

    Journal: Mediators of Inflammation

    Article Title: Alliin, a Garlic (Allium sativum) Compound, Prevents LPS-Induced Inflammation in 3T3-L1 Adipocytes

    doi: 10.1155/2013/381815

    Figure Lengend Snippet: Levels of phosphoextracellular signal-regulated kinase (ERK1/2 p44/p42) in mouse 3T3-L1 adipocytes. Cells were pretreated for 24 h with alliin 0.1 mM and subsequently exposed to 100 ng/mL of lipopolysaccharides (LPS) for 1 h afterward. (a) Representative Western blot with phospho-ERK1/2 and ERK1/2 antibodies; (b) protein levels of phospho-ERK1/2 and ERK1/2 in total cell extracts. CT control; AU arbitrary units. Data are expressed as mean ± standard deviations (SD) of three independent experiments * P ≤ 0.05; ** P ≤ 0.01.

    Article Snippet: The PVDF membranes were incubated overnight at 4°C with monoclonal antibodies against total and phosphorylated ERK1/2 (Cell Signaling and Santa Cruz Biotechnology, Santa Cruz, CA, USA), which were diluted 1 : 1,000 in blocking buffer.

    Techniques: Western Blot

    A proposal of how alliin could counteract the inflammatory state promoted by lipopolysaccharides (LPS) in 3T3-L1 adipocytes. (a) Activation of proinflammatory signaling pathway by lipopolysaccharides (LPS). (b) Alliin could reduce the Toll-like receptor-4 (TLR-4) pathway, possibly by diminishing the expression of related genes and proteins such as interleukin-6 (IL-6), monocyte chemostatic protein-1 (MCP-1), and early growth receptor-1 (Egr-1) and therefore regulates extracellular signal-regulated kinase (ERK1/2) activity.

    Journal: Mediators of Inflammation

    Article Title: Alliin, a Garlic (Allium sativum) Compound, Prevents LPS-Induced Inflammation in 3T3-L1 Adipocytes

    doi: 10.1155/2013/381815

    Figure Lengend Snippet: A proposal of how alliin could counteract the inflammatory state promoted by lipopolysaccharides (LPS) in 3T3-L1 adipocytes. (a) Activation of proinflammatory signaling pathway by lipopolysaccharides (LPS). (b) Alliin could reduce the Toll-like receptor-4 (TLR-4) pathway, possibly by diminishing the expression of related genes and proteins such as interleukin-6 (IL-6), monocyte chemostatic protein-1 (MCP-1), and early growth receptor-1 (Egr-1) and therefore regulates extracellular signal-regulated kinase (ERK1/2) activity.

    Article Snippet: The PVDF membranes were incubated overnight at 4°C with monoclonal antibodies against total and phosphorylated ERK1/2 (Cell Signaling and Santa Cruz Biotechnology, Santa Cruz, CA, USA), which were diluted 1 : 1,000 in blocking buffer.

    Techniques: Activation Assay, Expressing, Activity Assay

    The involvement of p38/GSK3β/Nrf-2 axis for cytoprotective effects of PGC-1α. To understand the molecular mechanism for cytoprotective effects of PGC-1α, the activation of Keap1-, GSK3β- or Hrd1 ( A ) and MAPKs ( B ), as an upstream signal molecules of Nrf-2, were compared to Mock and PGC-1α cells for indicated time points after H 2 O 2 treatment. To further elucidate the specificity of p38-mediated signal cascade for cytoprotective effects of PGC-1α cells, p38 specific effects were analyzed by western blotting ( C ) and MTT assay ( D ) in PGC-1α cells treated with H 2 O 2 in the presence or absence of p38 inhibitor (SB203580) for 2 h or 4 h, respectively. Inhibitor of ERK1/2 (PD98059) was used as non-effective control on PGC-1α effect. Activation of p38 or inactivation of GSK3β was checked with phosphor specific antibody at Thr180/Tyr182 residue or at Ser9 residue, respectively. Full-length blots of each tested protein are reported in Supplementary Figure S7 . Error bars denote the mean ± S.D. of triplicate samples. MTT assay was performed to n = 7. * p

    Journal: Scientific Reports

    Article Title: PGC-1α attenuates hydrogen peroxide-induced apoptotic cell death by upregulating Nrf-2 via GSK3β inactivation mediated by activated p38 in HK-2 Cells

    doi: 10.1038/s41598-017-04593-w

    Figure Lengend Snippet: The involvement of p38/GSK3β/Nrf-2 axis for cytoprotective effects of PGC-1α. To understand the molecular mechanism for cytoprotective effects of PGC-1α, the activation of Keap1-, GSK3β- or Hrd1 ( A ) and MAPKs ( B ), as an upstream signal molecules of Nrf-2, were compared to Mock and PGC-1α cells for indicated time points after H 2 O 2 treatment. To further elucidate the specificity of p38-mediated signal cascade for cytoprotective effects of PGC-1α cells, p38 specific effects were analyzed by western blotting ( C ) and MTT assay ( D ) in PGC-1α cells treated with H 2 O 2 in the presence or absence of p38 inhibitor (SB203580) for 2 h or 4 h, respectively. Inhibitor of ERK1/2 (PD98059) was used as non-effective control on PGC-1α effect. Activation of p38 or inactivation of GSK3β was checked with phosphor specific antibody at Thr180/Tyr182 residue or at Ser9 residue, respectively. Full-length blots of each tested protein are reported in Supplementary Figure S7 . Error bars denote the mean ± S.D. of triplicate samples. MTT assay was performed to n = 7. * p

    Article Snippet: Antibodies against caspase 3, cleaved caspase 3, Bax, Bcl2, phosphor-p53, total-p53, phosphor-GSK3β, totoal-GSK3β, phosphor-p38, phosphor-ERK1/2, phosphor-JNK, total-p38, total-ERK1/2, total-JNK, HO-1 and c-myc were all purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Pyrolysis Gas Chromatography, Activation Assay, Western Blot, MTT Assay

    ( A ) Phenobarbital and phenytoin decrease mRNA levels for BDNF and NT-3 and decrease phosphorylation of c-RAF, ERK1/2, and AKT in the neonatal brain. P7 pups received i.p. injection of phenobarbital (50 mg/kg), phenytoin (40 mg/kg), or vehicle (Co). Brain tissue from the thalamus was dissected at the various times indicated. Decreased density of the BDNF- and NT-3-specific bands is evident at 6, 12, and 24 h after administration of AEDs. Immunoblotting was performed with antiphospho-RAF, antiphospho-ERK1/2, antiphospho-AKT, ERK1/2 (phosphorylation state independent), or AKT (phosphorylation state independent) antibodies. There is a decrease in the levels of p-RAF, p-ERK1/2, and p-AKT at 6 h after injection of AEDs, whereas ERK1/2 and AKT (phosphorylation independent) are unaffected. ( B ) Quantitation of suppression by AEDs of mRNA levels for BDNF and NT-3 in the thalamus of infant rats. P7 pups received injection of phenobarbital (50 mg/kg, n = 9), phenytoin (50 mg/kg, n = 9), valproate (200 mg/kg, n = 9), or vehicle ( n = 9) and were killed at 6, 12, or 24 h after treatment ( n = 3 per group). mRNA levels for BDNF, NT-3, and β-actin were analyzed by means of PAGE and densitometrically quantitated. Values represent mean normalized ratios of the BDNF and NT-3 bands to β-actin ( n = 3 per point ± SEM). ANOVA revealed that there was a significant effect of treatment with AEDs on BDNF [ F (1,12) valproate = 29.79, P

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

    Article Title: Antiepileptic drugs and apoptotic neurodegeneration in the developing brain

    doi: 10.1073/pnas.222550499

    Figure Lengend Snippet: ( A ) Phenobarbital and phenytoin decrease mRNA levels for BDNF and NT-3 and decrease phosphorylation of c-RAF, ERK1/2, and AKT in the neonatal brain. P7 pups received i.p. injection of phenobarbital (50 mg/kg), phenytoin (40 mg/kg), or vehicle (Co). Brain tissue from the thalamus was dissected at the various times indicated. Decreased density of the BDNF- and NT-3-specific bands is evident at 6, 12, and 24 h after administration of AEDs. Immunoblotting was performed with antiphospho-RAF, antiphospho-ERK1/2, antiphospho-AKT, ERK1/2 (phosphorylation state independent), or AKT (phosphorylation state independent) antibodies. There is a decrease in the levels of p-RAF, p-ERK1/2, and p-AKT at 6 h after injection of AEDs, whereas ERK1/2 and AKT (phosphorylation independent) are unaffected. ( B ) Quantitation of suppression by AEDs of mRNA levels for BDNF and NT-3 in the thalamus of infant rats. P7 pups received injection of phenobarbital (50 mg/kg, n = 9), phenytoin (50 mg/kg, n = 9), valproate (200 mg/kg, n = 9), or vehicle ( n = 9) and were killed at 6, 12, or 24 h after treatment ( n = 3 per group). mRNA levels for BDNF, NT-3, and β-actin were analyzed by means of PAGE and densitometrically quantitated. Values represent mean normalized ratios of the BDNF and NT-3 bands to β-actin ( n = 3 per point ± SEM). ANOVA revealed that there was a significant effect of treatment with AEDs on BDNF [ F (1,12) valproate = 29.79, P

    Article Snippet: The membranes were incubated overnight at 4°C with the following antibodies: antiphospho-raf (1:2,000), antiphospho-ERK1/2 (1:2,000), antiphospho-AKT (1:2,000), anti-ERK1/2 phosphorylation state independent (1:2,000), or anti-AKT phosphorylation state independent (1:2,000) (Cell Signaling Technology, Beverly, MA).

    Techniques: Injection, Quantitation Assay, Polyacrylamide Gel Electrophoresis

    ERK 1/2 is implicated in 5FU toxicity on endothelial cells. (A) Representative images and percentage of EA.hy926 cells stained for SA β‐gal after the following treatments: no treatment (CTR); exposure to 5FU; pre‐incubation with GLP‐1 followed by exposure to 5FU; transfection with ERK1/2 siRNA (siERK), pre‐incubation with GLP‐1 and then exposure to 5FU; transfection with sham siRNA (sham); transfection with siERK. Magnification of pictures is 200×, and bars correspond to 50 μm. (B, C) Representative western blots for ERK1/2 and eNOS (B) and optical density (OD) of the protein bands normalized to that of actin (C) after the treatments described in (A). Graphs show mean ± SEM of five independent experiments. Comparisons were made by means of ANOVA followed by post hoc Tukey's multiple comparisons test. * Statistically significant versus CTR, # statistically significant versus 5FU and ¤ statistically significant versus siERK + GLP‐1 + FU.

    Journal: British Journal of Pharmacology

    Article Title: 5‐fluorouracil causes endothelial cell senescence: potential protective role of glucagon‐like peptide 1) 5‐fluorouracil causes endothelial cell senescence: potential protective role of glucagon‐like peptide 1

    doi: 10.1111/bph.13725

    Figure Lengend Snippet: ERK 1/2 is implicated in 5FU toxicity on endothelial cells. (A) Representative images and percentage of EA.hy926 cells stained for SA β‐gal after the following treatments: no treatment (CTR); exposure to 5FU; pre‐incubation with GLP‐1 followed by exposure to 5FU; transfection with ERK1/2 siRNA (siERK), pre‐incubation with GLP‐1 and then exposure to 5FU; transfection with sham siRNA (sham); transfection with siERK. Magnification of pictures is 200×, and bars correspond to 50 μm. (B, C) Representative western blots for ERK1/2 and eNOS (B) and optical density (OD) of the protein bands normalized to that of actin (C) after the treatments described in (A). Graphs show mean ± SEM of five independent experiments. Comparisons were made by means of ANOVA followed by post hoc Tukey's multiple comparisons test. * Statistically significant versus CTR, # statistically significant versus 5FU and ¤ statistically significant versus siERK + GLP‐1 + FU.

    Article Snippet: ERK 1/2 expression was silenced by means of ERK1/2 siRNA (Cell Signaling Technology, Danvers, MA, USA) transfected with Lipofectamine RNAiMAX Reagent 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol (final siRNA concentration 100 nmol·L−1 ); a sham siRNA (Cell Signaling Technology) was used as control.

    Techniques: Staining, Incubation, Transfection, Western Blot

    ERK1/2 activation was enhanced by S1P in LPS-challenged cells. The cells were incubated with inhibitory reagent against ERK1/2 (U0126) prior to LPS and S1P challenge. The induction of IL-6 (A) and IL-8 (B) was determined in culture supernatants by Luminex technology. The level of phosphorylated ERK1/2 was determined by FACE (Active Motif), as described in the Material and methods section, in cells following LPS or S1P/LPS challenge (C). Data are presented as means ± SD of triplicate determinations. Statistically significant ( p

    Journal: European journal of immunology

    Article Title: TLR4 and S1P receptors cooperate to enhance inflammatory cytokine production in human gingival epithelial cells

    doi: 10.1002/eji.200737898

    Figure Lengend Snippet: ERK1/2 activation was enhanced by S1P in LPS-challenged cells. The cells were incubated with inhibitory reagent against ERK1/2 (U0126) prior to LPS and S1P challenge. The induction of IL-6 (A) and IL-8 (B) was determined in culture supernatants by Luminex technology. The level of phosphorylated ERK1/2 was determined by FACE (Active Motif), as described in the Material and methods section, in cells following LPS or S1P/LPS challenge (C). Data are presented as means ± SD of triplicate determinations. Statistically significant ( p

    Article Snippet: To examine the effect of ERK1/2, cells were pretreated with the ERK1/2 inhibitor, U0126 (25 μM; Cell Signal), for 2 h prior to LPS and S1P stimulation.

    Techniques: Activation Assay, Incubation, Luminex

    Reduction of Enah downregulates p-Erk1/2, p-AKT, and p-p65 and inhibits EMT (epithelial-mesenchymal transition) progress in GC cells. a Decreased protein levels of p-Erk1/2, p-AKT, p-p65, Vimentin, Fibronectin, and increased protein level of E-cadherin in LV-shEnah cells compared with LV-shcontrol cells. No significant difference was detected in the protein expression of p-STAT3. b Morphology of MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells as visualized using phase-contrast microscopy (magnification × 200). c Immunofluorescence analysis of E-cadherin and Vimentin expression in MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells. * p

    Journal: Cell Death & Disease

    Article Title: Enah overexpression is correlated with poor survival and aggressive phenotype in gastric cancer

    doi: 10.1038/s41419-018-1031-x

    Figure Lengend Snippet: Reduction of Enah downregulates p-Erk1/2, p-AKT, and p-p65 and inhibits EMT (epithelial-mesenchymal transition) progress in GC cells. a Decreased protein levels of p-Erk1/2, p-AKT, p-p65, Vimentin, Fibronectin, and increased protein level of E-cadherin in LV-shEnah cells compared with LV-shcontrol cells. No significant difference was detected in the protein expression of p-STAT3. b Morphology of MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells as visualized using phase-contrast microscopy (magnification × 200). c Immunofluorescence analysis of E-cadherin and Vimentin expression in MKN45-shcontrol, MKN45-shEnah, AGS-shcontrol and AGS-shEnah cells. * p

    Article Snippet: The primary antibodies used in the experiment were as following: Enah (BD Bioscience; US); PCNA (Cell Signaling Technology, USA), p-Erk1/2/Erk1/2 (Cell Signaling Technology, USA), p-STAT/STAT (Cell Signaling Technology, USA), p-AKT/AKT (Cell Signaling Technology, USA), p-p65/p65 NF-κB (Cell Signaling Technology, USA), E-cadherin (Cell Signaling Technology, USA),Vimentin (Abcam, USA), Fibronectin (Santa Cruz Biotechnology, USA) and β-actin (Sigma, USA).

    Techniques: Expressing, Microscopy, Immunofluorescence

    Fasudil did not affect the LPS-stimulated phosphorylation of ERK1/2 and JNK in RMG cells. A : The protein levels of p44/42 (Erk1/2), phospho-ERK1/2 (p-ERK1/2), c-Jun N-terminal kinase (JNK), and phospho-JNK (p-JNK) in lipopolysaccharide (LPS)-stimulated and/or fasudil-treated retinal microglial (RMG) cells. B : The relative protein band intensities were quantified with densitometric analyses and normalized to β-actin, p-JNK, JNK, p-ERK1/2, and ERK1/2. Values represent the means ± standard deviation (SD) of three independent experiments performed in triplicate. **p

    Journal: Molecular Vision

    Article Title: Fasudil inhibits LPS-induced migration of retinal microglial cells via regulating p38-MAPK signaling pathway

    doi:

    Figure Lengend Snippet: Fasudil did not affect the LPS-stimulated phosphorylation of ERK1/2 and JNK in RMG cells. A : The protein levels of p44/42 (Erk1/2), phospho-ERK1/2 (p-ERK1/2), c-Jun N-terminal kinase (JNK), and phospho-JNK (p-JNK) in lipopolysaccharide (LPS)-stimulated and/or fasudil-treated retinal microglial (RMG) cells. B : The relative protein band intensities were quantified with densitometric analyses and normalized to β-actin, p-JNK, JNK, p-ERK1/2, and ERK1/2. Values represent the means ± standard deviation (SD) of three independent experiments performed in triplicate. **p

    Article Snippet: The membranes were subsequently blocked with the use of 5% milk in Tris Buffered saline Tween (TBST; Beyotime Institute of Biotechnology) for 1 h and incubated overnight with antibodies against p38 (Cell Signaling Technology, Beverly, MA), p-p38 (Cell Signaling Technology), p44/42 (Erk1/2) (Cell Signaling Technology), phospho-ERK1/2 (p-ERK1/2) (Cell Signaling Technology), c-Jun N-terminal kinase (JNK; Cell Signaling Technology), phospho-JNK (p-JNK; Cell Signaling Technology), MMP-2 (Abcam), MMP-9 (Abcam) or β-actin (Abcam) in TBST containing 5% defatted milk at 4 ºC followed by incubation with horseradish peroxidase-linked secondary antibodies (Abcam) at 4 ºC for additional 1 h. The immunobands were detected with an enhanced chemiluminescence kit (Amersham, ECL Plus, Freiburg, Germany), and the intensity was measured using ImageJ software (NIH, Bethesda, MD).

    Techniques: Standard Deviation

    MKP3 is dependent on ERK1/2 but not Akt activation . (A) Beads soaked with specific inhibitors were placed in the caudal hindbrain or in the presumptive MHB of HH7 + -8 explanted embryos. Pharmacological treatments were as follows: DMSO (a), FGFR inhibitor SU5402 (b), ERK1/2 inhibitor PD184352 (c), PI3K inhibitor LY294002 (d). Explants were incubated during 6 h at 38°C. (B) Jurkat cells (left side lanes) and HH7-8 explanted embryos (right side lanes) were treated with DMSO, SU5402, PD184352 or LY294002 and analyzed by western blot for total and phosphorylated forms of Akt and ERK1/2. PD184352 treatment impeded ERK1/2 phosphorylation without affecting the PI3K-Akt pathway and, conversely, LY294002 treatment abolished Akt phosphorylation without affecting the Ras-ERK1/2 pathway. Embryos are shown in whole-mount with anterior to the top.

    Journal: BMC Developmental Biology

    Article Title: FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

    doi: 10.1186/1471-213X-9-61

    Figure Lengend Snippet: MKP3 is dependent on ERK1/2 but not Akt activation . (A) Beads soaked with specific inhibitors were placed in the caudal hindbrain or in the presumptive MHB of HH7 + -8 explanted embryos. Pharmacological treatments were as follows: DMSO (a), FGFR inhibitor SU5402 (b), ERK1/2 inhibitor PD184352 (c), PI3K inhibitor LY294002 (d). Explants were incubated during 6 h at 38°C. (B) Jurkat cells (left side lanes) and HH7-8 explanted embryos (right side lanes) were treated with DMSO, SU5402, PD184352 or LY294002 and analyzed by western blot for total and phosphorylated forms of Akt and ERK1/2. PD184352 treatment impeded ERK1/2 phosphorylation without affecting the PI3K-Akt pathway and, conversely, LY294002 treatment abolished Akt phosphorylation without affecting the Ras-ERK1/2 pathway. Embryos are shown in whole-mount with anterior to the top.

    Article Snippet: Afterwards, they were treated with 6% H2 O2 /PBTx (PBS 1%triton) 2 h at rt, washed in PBTx, incubated in blocking solution (PBTx 10% heat inactivated goat serum) for 1 h and subsequently incubated either with polyclonal antibody anti-GFP [1:500] (Molecular Probes) overnight at 4°C [ ], or anti-dual phosphorylated (dp)ERK1/2 [1:50] (Cell Signaling) during 5 days at 4°C.

    Techniques: Activation Assay, Incubation, Western Blot

    Model depicting the cooperation of vHnf1 and FGF signals in the induction of MafB and Krox20 in the caudal hindbrain . (a) vHnf1 is very early expressed in the caudal neural plate with a sharp boundary laying in the prospective r4/r5 boundary. This expression may be initiated very early in response to RA from the axial and paraxial mesoderm during mid/late stages of gastrulation [ 12 , 64 , 65 ]. Anterior limit of expression of vHnf1 may be established by mutual repression with an irx gene [ 61 , 65 ], probably irx3 (unpublished results). vHnf1 rapidly induces Fgf3 in the caudal hindbrain [ 1 ] , which activates the Ras-ERK1/2 pathway. vHnf1 and Fgf3-ERK1/2 co-operate for the induction of MafB expression in r5 and r6 at 4-5ss and Krox20 in r5 at 6-7ss. Krox20 induction is probably also dependent on MafB expression as suggested in mouse and zebrafish [ 8 , 11 , 66 , 67 ]. Krox20 is initially expressed in a narrow domain caudal to r4 and subsequently expands its expression area due to non-cell autonomous induction. Coinciding with the onset of Krox20 in r5, vHnf1 progressively regresses to r6 between 7 and 10ss. Mutual repression between vHnf1 and Krox20 may prevent expansion of Krox20 to r6. Anterior is to the left. (b) Putative regulation of MafB and Krox20 expression by vHnf1 modulating FGF signaling through MKP3 .

    Journal: BMC Developmental Biology

    Article Title: FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

    doi: 10.1186/1471-213X-9-61

    Figure Lengend Snippet: Model depicting the cooperation of vHnf1 and FGF signals in the induction of MafB and Krox20 in the caudal hindbrain . (a) vHnf1 is very early expressed in the caudal neural plate with a sharp boundary laying in the prospective r4/r5 boundary. This expression may be initiated very early in response to RA from the axial and paraxial mesoderm during mid/late stages of gastrulation [ 12 , 64 , 65 ]. Anterior limit of expression of vHnf1 may be established by mutual repression with an irx gene [ 61 , 65 ], probably irx3 (unpublished results). vHnf1 rapidly induces Fgf3 in the caudal hindbrain [ 1 ] , which activates the Ras-ERK1/2 pathway. vHnf1 and Fgf3-ERK1/2 co-operate for the induction of MafB expression in r5 and r6 at 4-5ss and Krox20 in r5 at 6-7ss. Krox20 induction is probably also dependent on MafB expression as suggested in mouse and zebrafish [ 8 , 11 , 66 , 67 ]. Krox20 is initially expressed in a narrow domain caudal to r4 and subsequently expands its expression area due to non-cell autonomous induction. Coinciding with the onset of Krox20 in r5, vHnf1 progressively regresses to r6 between 7 and 10ss. Mutual repression between vHnf1 and Krox20 may prevent expansion of Krox20 to r6. Anterior is to the left. (b) Putative regulation of MafB and Krox20 expression by vHnf1 modulating FGF signaling through MKP3 .

    Article Snippet: Afterwards, they were treated with 6% H2 O2 /PBTx (PBS 1%triton) 2 h at rt, washed in PBTx, incubated in blocking solution (PBTx 10% heat inactivated goat serum) for 1 h and subsequently incubated either with polyclonal antibody anti-GFP [1:500] (Molecular Probes) overnight at 4°C [ ], or anti-dual phosphorylated (dp)ERK1/2 [1:50] (Cell Signaling) during 5 days at 4°C.

    Techniques: Expressing

    Phosphorylated forms of Akt and ERK1/2 are present in the caudal hindbrain . (a) Western Blot analysis of protein extracts of whole embryos (left column) and isolated caudal hindbrain (right column). Phosphorylated (activated) forms of both Akt and ERK1/2 were present in protein extracts from the caudal hindbrain. Total forms of Akt and ERK1/2 were used as loading controls. pERK1/2 immunodetection is shown in red (b, d-e, h-k) or in green fluorescence in (f). In situ hybridization with vHnf1-Hoxb1 (c) and vHnf1 (g) to position the caudal hindbrain. (i-k) Control experiments to show the specificity of pErk staining: embryos incubated with DMSO (i) or with SU5402 (j), and assayed for pErk (note that specific pErk staining in the MHB and in the caudal hindbrain disappears upon SU5402 treatment); (k) embryos treated as in (i) but without pErk-Ab incubation, as negative control. Whole-mount embryos (b-d, f-g, i-k), or flat-mounted hindbrains (e, h) at indicated stages. ANR, anterior neural ridge; cHB, caudal hindbrain; FB, forebrain; HB, hindbrain; im, intermediate mesoderm; MHB, midbrain-hindbrain boundary; pm, precardiac mesoderm; ps: primitive strike; psm, presomitic mesoderm. Anterior is at the top.

    Journal: BMC Developmental Biology

    Article Title: FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

    doi: 10.1186/1471-213X-9-61

    Figure Lengend Snippet: Phosphorylated forms of Akt and ERK1/2 are present in the caudal hindbrain . (a) Western Blot analysis of protein extracts of whole embryos (left column) and isolated caudal hindbrain (right column). Phosphorylated (activated) forms of both Akt and ERK1/2 were present in protein extracts from the caudal hindbrain. Total forms of Akt and ERK1/2 were used as loading controls. pERK1/2 immunodetection is shown in red (b, d-e, h-k) or in green fluorescence in (f). In situ hybridization with vHnf1-Hoxb1 (c) and vHnf1 (g) to position the caudal hindbrain. (i-k) Control experiments to show the specificity of pErk staining: embryos incubated with DMSO (i) or with SU5402 (j), and assayed for pErk (note that specific pErk staining in the MHB and in the caudal hindbrain disappears upon SU5402 treatment); (k) embryos treated as in (i) but without pErk-Ab incubation, as negative control. Whole-mount embryos (b-d, f-g, i-k), or flat-mounted hindbrains (e, h) at indicated stages. ANR, anterior neural ridge; cHB, caudal hindbrain; FB, forebrain; HB, hindbrain; im, intermediate mesoderm; MHB, midbrain-hindbrain boundary; pm, precardiac mesoderm; ps: primitive strike; psm, presomitic mesoderm. Anterior is at the top.

    Article Snippet: Afterwards, they were treated with 6% H2 O2 /PBTx (PBS 1%triton) 2 h at rt, washed in PBTx, incubated in blocking solution (PBTx 10% heat inactivated goat serum) for 1 h and subsequently incubated either with polyclonal antibody anti-GFP [1:500] (Molecular Probes) overnight at 4°C [ ], or anti-dual phosphorylated (dp)ERK1/2 [1:50] (Cell Signaling) during 5 days at 4°C.

    Techniques: Western Blot, Isolation, Immunodetection, Fluorescence, In Situ Hybridization, Staining, Incubation, Negative Control

    MafB and Krox20 are dependent on ERK1/2 but not on Akt activation . HH7 + -8 embryos (a-d, f-i,k-m) or HH9 (e, j) were explanted and beads soaked in specific inhibitors were placed in the caudal hindbrain. Afterwards, embryos were analyzed for Krox20 , MafB or Fgf3 expression. Pharmacological treatments were as follows: DMSO (a, f,k), FGFR inhibitor SU5402 (b, e,g,j,l), ERK1/2 inhibitor PD184352 (c, h,m), or PI3K inhibitor LY294002 (d, i), during 6 h. Embryos are shown in whole-mount with anterior to the top.

    Journal: BMC Developmental Biology

    Article Title: FGF signaling controls caudal hindbrain specification through Ras-ERK1/2 pathway

    doi: 10.1186/1471-213X-9-61

    Figure Lengend Snippet: MafB and Krox20 are dependent on ERK1/2 but not on Akt activation . HH7 + -8 embryos (a-d, f-i,k-m) or HH9 (e, j) were explanted and beads soaked in specific inhibitors were placed in the caudal hindbrain. Afterwards, embryos were analyzed for Krox20 , MafB or Fgf3 expression. Pharmacological treatments were as follows: DMSO (a, f,k), FGFR inhibitor SU5402 (b, e,g,j,l), ERK1/2 inhibitor PD184352 (c, h,m), or PI3K inhibitor LY294002 (d, i), during 6 h. Embryos are shown in whole-mount with anterior to the top.

    Article Snippet: Afterwards, they were treated with 6% H2 O2 /PBTx (PBS 1%triton) 2 h at rt, washed in PBTx, incubated in blocking solution (PBTx 10% heat inactivated goat serum) for 1 h and subsequently incubated either with polyclonal antibody anti-GFP [1:500] (Molecular Probes) overnight at 4°C [ ], or anti-dual phosphorylated (dp)ERK1/2 [1:50] (Cell Signaling) during 5 days at 4°C.

    Techniques: Activation Assay, Expressing

    Effect of depleting GGA1 and GGA2 on α 2B -AR-mediated signaling. ( A ) HEK293 cells were transfected with control shRNA or individual GGA shRNA for 36 h. After starvation for 3 h, the cells were stimulated with UK14304 at the concentration of 1 μM for 5 min at 37 °C. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel is a representative blot of ERK1/2 activation and lower panel shows total ERK1/2 expression. ( B ) Quantitative data shown in A). The data shown are percentages of the mean value obtained from cells transfected with control shRNA and are presented as the mean ± S.E. of at least three experiments. * p

    Journal: Scientific Reports

    Article Title: Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2

    doi: 10.1038/srep37921

    Figure Lengend Snippet: Effect of depleting GGA1 and GGA2 on α 2B -AR-mediated signaling. ( A ) HEK293 cells were transfected with control shRNA or individual GGA shRNA for 36 h. After starvation for 3 h, the cells were stimulated with UK14304 at the concentration of 1 μM for 5 min at 37 °C. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel is a representative blot of ERK1/2 activation and lower panel shows total ERK1/2 expression. ( B ) Quantitative data shown in A). The data shown are percentages of the mean value obtained from cells transfected with control shRNA and are presented as the mean ± S.E. of at least three experiments. * p

    Article Snippet: Anti-ERK antibodies detecting total ERK1/2 expression and GM130 were from Cell Signaling Technology, Inc. (Beverly, MA).

    Techniques: Transfection, shRNA, Concentration Assay, Activation Assay, Western Blot, Expressing

    The expression of inflammatory cytokines in response to Ang II with or without the presence of MAPK and NF-κB inhibitors. mRNA expression of IL-8, MCP-1 and IL-6 was measured by real-time PCR 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580 ( A ) ERK1/2 inhibitor PD98059 ( B ) JNK inhibitor SP600125 ( C ) and NF-κB inhibitor BAY11-7082 ( D ) all at a final concentration of 10 μM. ( E ) The protein expression levels of IL-8, MCP-1 and IL-6 were measured by ELISA 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580, ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and NF-κB inhibitor BAY11-7082, all at a final concentration of 10 μM. The data were shown as mean ± SEM (* p

    Journal: Scientific Reports

    Article Title: Downregulating p22phox ameliorates inflammatory response in Angiotensin II-induced oxidative stress by regulating MAPK and NF-κB pathways in ARPE-19 cells

    doi: 10.1038/srep14362

    Figure Lengend Snippet: The expression of inflammatory cytokines in response to Ang II with or without the presence of MAPK and NF-κB inhibitors. mRNA expression of IL-8, MCP-1 and IL-6 was measured by real-time PCR 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580 ( A ) ERK1/2 inhibitor PD98059 ( B ) JNK inhibitor SP600125 ( C ) and NF-κB inhibitor BAY11-7082 ( D ) all at a final concentration of 10 μM. ( E ) The protein expression levels of IL-8, MCP-1 and IL-6 were measured by ELISA 48 hours after stimulation by 10 −6 M Ang II with or without the presence of the p38 MAPK inhibitor SB203580, ERK1/2 inhibitor PD98059, JNK inhibitor SP600125 and NF-κB inhibitor BAY11-7082, all at a final concentration of 10 μM. The data were shown as mean ± SEM (* p

    Article Snippet: After being serum starved for 24 hours, cells were pre-incubated with p38MAPK inhibitor SB203580 (Cell Signaling Technology, Danvers, MA), JNK inhibitor SP600125 (Cell Signaling Technology, Danvers, MA), ERK1/2 inhibitor PD98059 (Cell Signaling Technology, Danvers, MA) and NF-κB inhibitor BAY11-7082 (Sigma-Aldrich, St. Louis, MO) at the concentration of 10 μM for 30 minutes.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Recombinant human (rh) RELM-β has mitogenic effects on human lung microvascular endothelial cells (HMVEC-Ls) and human pulmonary artery smooth muscle cells (HPASMCs) and activates ERK1/2. HMVEC-Ls ( A ) and HPASMCs ( B ) were serum and growth factor

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: Resistin-Like Molecule-? in Scleroderma-Associated Pulmonary Hypertension

    doi: 10.1165/rcmb.2008-0271OC

    Figure Lengend Snippet: Recombinant human (rh) RELM-β has mitogenic effects on human lung microvascular endothelial cells (HMVEC-Ls) and human pulmonary artery smooth muscle cells (HPASMCs) and activates ERK1/2. HMVEC-Ls ( A ) and HPASMCs ( B ) were serum and growth factor

    Article Snippet: To determine if RELM-β–induced mitogenesis was ERK1/2-dependent, HMVEC-Ls or HPASMCs that were prepared as stated above were preincubated for 1 hour with U0126 (10 μM), a pharmacologic ERK1/2 inhibitor (Cell Signaling Technologies).

    Techniques: Recombinant

    Ciprofloxacin induces Erk1/2 activation and has no effect on TGFβ/Smad signaling. (A) Cells were treated with 50 μ g/ml of ciprofloxacin and P-Erk1/2 levels were analyzed after 12 h; bar graph on the right represents relative quantification of 4 experiments. (B) Cells were pretreated with Erk inhibitor UO126 and mRNA levels of MMP1 were analyzed after 48 h of ciprofloxacin treatment (50 μ g/ml) (n=3). * P≤0.05 and ** P≤0.01. (C) Healthy cells and systemic sclerosis dermal fibroblasts were pretreated with 50 μ g/ml of ciprofloxacin overnight then with 2.5 ng/ml of TGFβ and levels of P-Smad3, total Smad2/3, P-Smad1 and total Smad1 were analyzed by western blot analysis. Representative blots from 3 experiments are shown. Cx, ciprofloxacin; NS, healthy cells; SSc, systemic sclerosis; c, control.

    Journal: International Journal of Molecular Medicine

    Article Title: Ciprofloxacin has antifibrotic effects in scleroderma fibroblasts via downregulation of Dnmt1 and upregulation of Fli1

    doi: 10.3892/ijmm.2012.1150

    Figure Lengend Snippet: Ciprofloxacin induces Erk1/2 activation and has no effect on TGFβ/Smad signaling. (A) Cells were treated with 50 μ g/ml of ciprofloxacin and P-Erk1/2 levels were analyzed after 12 h; bar graph on the right represents relative quantification of 4 experiments. (B) Cells were pretreated with Erk inhibitor UO126 and mRNA levels of MMP1 were analyzed after 48 h of ciprofloxacin treatment (50 μ g/ml) (n=3). * P≤0.05 and ** P≤0.01. (C) Healthy cells and systemic sclerosis dermal fibroblasts were pretreated with 50 μ g/ml of ciprofloxacin overnight then with 2.5 ng/ml of TGFβ and levels of P-Smad3, total Smad2/3, P-Smad1 and total Smad1 were analyzed by western blot analysis. Representative blots from 3 experiments are shown. Cx, ciprofloxacin; NS, healthy cells; SSc, systemic sclerosis; c, control.

    Article Snippet: The ERK1/2 inhibitor UO126 was purchased from Cell Signaling Technology, Inc.

    Techniques: Activation Assay, Western Blot

    PLX8394 effectively inhibits ERK1/2 signaling in patient tumors comparable to dabrafenib/trametinib treatment A. H E and IHC analysis of pERK1/2 staining from a representative mutant BRAF patient sample (TJU-MEL-27A) treated with either DMSO, vemurafenib (1 μM), combo (1 μM dabrafenib/16 nM trametinib) or PLX8394 (0.5 μM). B. Quantitation of A across a panel of 6 different mutant BRAF melanoma patient samples. C. Western blot analysis of ERK1/2 signaling and PARP cleavage from a representative patient sample (TJU-MEL-27A). D. Western blot quantitation of the normalized pERK1/2 to ERK2 signal from 5 patient samples. E. RPPA data from mutant BRAF patient samples were analyzed via GSEA. Patient explants treated with vemurafenib (left) and PLX8394 (right), were grouped and compared to DMSO treated samples. Enrichment of the Programmed Cell Death/Apoptosis and Cell Cycle Arrest GO pathways and corresponding changes in RPPA determined protein levels compared to DMSO are shown. Pathway nodes and protein levels for all treatments are on the same scale (bottom left).

    Journal: Molecular cancer therapeutics

    Article Title: Response and resistance to paradox breaking BRAF inhibitor in melanomas in vivo and ex vivo

    doi: 10.1158/1535-7163.MCT-17-0705

    Figure Lengend Snippet: PLX8394 effectively inhibits ERK1/2 signaling in patient tumors comparable to dabrafenib/trametinib treatment A. H E and IHC analysis of pERK1/2 staining from a representative mutant BRAF patient sample (TJU-MEL-27A) treated with either DMSO, vemurafenib (1 μM), combo (1 μM dabrafenib/16 nM trametinib) or PLX8394 (0.5 μM). B. Quantitation of A across a panel of 6 different mutant BRAF melanoma patient samples. C. Western blot analysis of ERK1/2 signaling and PARP cleavage from a representative patient sample (TJU-MEL-27A). D. Western blot quantitation of the normalized pERK1/2 to ERK2 signal from 5 patient samples. E. RPPA data from mutant BRAF patient samples were analyzed via GSEA. Patient explants treated with vemurafenib (left) and PLX8394 (right), were grouped and compared to DMSO treated samples. Enrichment of the Programmed Cell Death/Apoptosis and Cell Cycle Arrest GO pathways and corresponding changes in RPPA determined protein levels compared to DMSO are shown. Pathway nodes and protein levels for all treatments are on the same scale (bottom left).

    Article Snippet: By western blot analysis, vemurafenib inhibition of ERK1/2 phosphorylation was again variable, but statistically significant compared to vehicle treatment ( & ; ).

    Techniques: Immunohistochemistry, Staining, Mutagenesis, Quantitation Assay, Western Blot

    PLX8394 suppresses MEK/ERK signaling in mutant BRAF splice variant expressing cells and is associated with reduction of splice variant homodimerization A. 1205LuTR GAL4-ELK1 cells were used to generate xenograft tumors that were harvested and dissected into ~1 mm 3 pieces for use in explant system. After 48 hours of treatment, lysates were collected and analyzed by western blotting. Using densitometry, the normalized ratio of phospho ERK1/2 to ERK2 levels and cleaved PARP to HSP90 for each cohort was quantified and graphed. Data was analyzed with a two-way ANOVA corrected for multiple comparisons with Tukey analysis. Error bars are +/− SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. B. Similar to A except PRT#3 cell line was used to generate xenograft tumors. C. Similar to A but PRT#4 xenograft tumors. D. Western blots of whole cell lysates from 1205LuTR FLAG/Myc BRAF ΔEx 2-8 cells after 48 hours of doxycycline induced splice variant expression and an additional 4 hours of PLX4720 or PLX8394 treatment at the indicated concentration. E. Parallel lysates from D were used to immunoprecipitate the FLAG tagged mutant BRAF splice variant and western blots reveal its associated Myc tagged binding partner. F. Quantification of splice variant homodimerization after treatment with 1 μM PLX4720 and 0.5 μM PLX8394 (N=4).

    Journal: Molecular cancer therapeutics

    Article Title: Response and resistance to paradox breaking BRAF inhibitor in melanomas in vivo and ex vivo

    doi: 10.1158/1535-7163.MCT-17-0705

    Figure Lengend Snippet: PLX8394 suppresses MEK/ERK signaling in mutant BRAF splice variant expressing cells and is associated with reduction of splice variant homodimerization A. 1205LuTR GAL4-ELK1 cells were used to generate xenograft tumors that were harvested and dissected into ~1 mm 3 pieces for use in explant system. After 48 hours of treatment, lysates were collected and analyzed by western blotting. Using densitometry, the normalized ratio of phospho ERK1/2 to ERK2 levels and cleaved PARP to HSP90 for each cohort was quantified and graphed. Data was analyzed with a two-way ANOVA corrected for multiple comparisons with Tukey analysis. Error bars are +/− SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. B. Similar to A except PRT#3 cell line was used to generate xenograft tumors. C. Similar to A but PRT#4 xenograft tumors. D. Western blots of whole cell lysates from 1205LuTR FLAG/Myc BRAF ΔEx 2-8 cells after 48 hours of doxycycline induced splice variant expression and an additional 4 hours of PLX4720 or PLX8394 treatment at the indicated concentration. E. Parallel lysates from D were used to immunoprecipitate the FLAG tagged mutant BRAF splice variant and western blots reveal its associated Myc tagged binding partner. F. Quantification of splice variant homodimerization after treatment with 1 μM PLX4720 and 0.5 μM PLX8394 (N=4).

    Article Snippet: By western blot analysis, vemurafenib inhibition of ERK1/2 phosphorylation was again variable, but statistically significant compared to vehicle treatment ( & ; ).

    Techniques: Mutagenesis, Variant Assay, Expressing, Western Blot, Concentration Assay, Binding Assay

    PLX8394 effectively reduces ERK1/2 signaling and tumor volume in vivo A. 1205LuTR GAL4-ELK1 cells were treated for 24 hours with either DMSO or 0.5 μM PLX8394. Lysates were obtained from three independent experiments and processed for RPPA analysis. A heat map was generated using median-centered data across each protein measurement for each sample. B. Proteins with a p value ≤ 0.01 and a fold change of ≥ 1.5 that were significantly altered following PLX8394 treatment. C. Mice bearing 1205LuTR GAL4-ELK1 xenografts were fed either vehicle chow or PLX8394 laced chow. Representative images of a vehicle and PLX8394 treated mouse with overlaid luciferase output across 10 days of treatment are shown. D. Quantification of firefly luciferase. Graph depicts fold change in luciferase output per tumor volume compared to vehicle for each day of treatment. E. Average fold change in tumor volume between mice fed vehicle chow and PLX8394-laced chow.

    Journal: Molecular cancer therapeutics

    Article Title: Response and resistance to paradox breaking BRAF inhibitor in melanomas in vivo and ex vivo

    doi: 10.1158/1535-7163.MCT-17-0705

    Figure Lengend Snippet: PLX8394 effectively reduces ERK1/2 signaling and tumor volume in vivo A. 1205LuTR GAL4-ELK1 cells were treated for 24 hours with either DMSO or 0.5 μM PLX8394. Lysates were obtained from three independent experiments and processed for RPPA analysis. A heat map was generated using median-centered data across each protein measurement for each sample. B. Proteins with a p value ≤ 0.01 and a fold change of ≥ 1.5 that were significantly altered following PLX8394 treatment. C. Mice bearing 1205LuTR GAL4-ELK1 xenografts were fed either vehicle chow or PLX8394 laced chow. Representative images of a vehicle and PLX8394 treated mouse with overlaid luciferase output across 10 days of treatment are shown. D. Quantification of firefly luciferase. Graph depicts fold change in luciferase output per tumor volume compared to vehicle for each day of treatment. E. Average fold change in tumor volume between mice fed vehicle chow and PLX8394-laced chow.

    Article Snippet: By western blot analysis, vemurafenib inhibition of ERK1/2 phosphorylation was again variable, but statistically significant compared to vehicle treatment ( & ; ).

    Techniques: In Vivo, Generated, Mouse Assay, Luciferase

    Effect of specific MAPK inhibitors on telomerase. For specific inhibition of p38 activation, 10 µM SB203580 or 20 µM SB202190, of JNK activation, 5 µM SP600125 or 20 µM JNK inhibitor V were used, respectively. 10 µM U0126 or 40 µM PB98059 were used for ERK1/2 inhibition. Cells were pre-treated with the inhibitors and subsequently exposed to MTBITC or the solvent control (0.1% DMSO). a) Immunoblot analysis shows effective inhibition of the target proteins. β-actin was used as loading control b) Pre-treated cells were analysed for full length hTERT mRNA expression after 1 h exposure to 25 µM MTBITC. PBDG was used as reference gene. mRNA expression was calculated relative to the corresponding solvent control (n = 3). c+d) Pre-treated cells were assayed for telomerase enzyme activity after 3 h (d) or 24 h (e) MTBITC exposure using the TRAP-ELISA assay. Telomerase activity was calculated relative to the corresponding solvent control; bars are mean ± SEM (n = 3). Inhibitors 3×: combination of SB203580, SP600125 and U0126.

    Journal: PLoS ONE

    Article Title: The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

    doi: 10.1371/journal.pone.0053240

    Figure Lengend Snippet: Effect of specific MAPK inhibitors on telomerase. For specific inhibition of p38 activation, 10 µM SB203580 or 20 µM SB202190, of JNK activation, 5 µM SP600125 or 20 µM JNK inhibitor V were used, respectively. 10 µM U0126 or 40 µM PB98059 were used for ERK1/2 inhibition. Cells were pre-treated with the inhibitors and subsequently exposed to MTBITC or the solvent control (0.1% DMSO). a) Immunoblot analysis shows effective inhibition of the target proteins. β-actin was used as loading control b) Pre-treated cells were analysed for full length hTERT mRNA expression after 1 h exposure to 25 µM MTBITC. PBDG was used as reference gene. mRNA expression was calculated relative to the corresponding solvent control (n = 3). c+d) Pre-treated cells were assayed for telomerase enzyme activity after 3 h (d) or 24 h (e) MTBITC exposure using the TRAP-ELISA assay. Telomerase activity was calculated relative to the corresponding solvent control; bars are mean ± SEM (n = 3). Inhibitors 3×: combination of SB203580, SP600125 and U0126.

    Article Snippet: The ERK1/2 inhibitor U0126, p38 inhibitor SB202190 and ERK1/2 inhibitor PB98059 were obtained from Cell Signalling (Boston, USA).

    Techniques: Inhibition, Activation Assay, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

    Antcin M activates Nrf2-dependent anti-oxidant defense in HNDFs A. To determine the free-radical scavenging effect of ANM, cell-free DPPH assay was performed. NAC and RES were used as positive controls. B. , C. To quantify the mRNA expression levels of HO-1 and NQO-1, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 12 h. Total RNA was extracted and subjected to Q-PCR analysis. Relative mRNA levels were normalized with β-actin mRNA. D. To determine the protein expression levels of HO-1, NQO-1 and Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) for 24 h. Total cell lysates were prepared and subjected to western blot analysis to monitor the expression levels of HO-1, NQO-1 and Nrf2. E. To determine the Nrf2 transcriptional activity, HNDFs were transiently transfected with ARE promoter construct using lipofectamine and incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 6 h. Cell lysates were mixed with luciferase reagents and quantified using an illuminometer. Relative ARE promoter activity was calculated by dividing the relative luciferace unit (RLU) of treated cells by RLU of untreated cells (NG). F. To determine the nuclear localization of Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence or HG (30 mM) for 2 h. The protein expression and localization of Nrf2 was measured by immunofluorescence using Nrf2 specific primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). The subcellular and nuclear localization of Nrf2 was photographed using a fluoroscence microscope. DAPI (1 μM) was used to stain the nucleus. G. HNDFs were pre-incubated with AKT, ERK1/2, JNK/SAPK and p38 MAPK inhbitors LY294002 (LY, 30 μM), PD98059 (PD, 30 μM), SP600125 (SP, 30 μM) and SB203580 (SB, 30 μM), respectively for 2 h and then incubated with ANM (10 μM) in the presence of HG (30 mM) 2 h. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis. GAPDH and histone H3 served as internal controls for the cytoplasmic and nuclear fraction, respectively. H. The Keap-1 protein expression level was determined by western blotting. I. Effect of ANM on ubiquitination of Keap-1. Equivalent amount of proteins were immune-precipitated with Keap-1 antibody and visualized by western blotting with ubiquitin antibody. Histogram shows the percentage of ubiquinated Keap-1. Results expressed as mean ± S.E.M of three indipendent expriments. Statistical significance was set at Ф P

    Journal: Oncotarget

    Article Title: A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1

    doi: 10.18632/oncotarget.11229

    Figure Lengend Snippet: Antcin M activates Nrf2-dependent anti-oxidant defense in HNDFs A. To determine the free-radical scavenging effect of ANM, cell-free DPPH assay was performed. NAC and RES were used as positive controls. B. , C. To quantify the mRNA expression levels of HO-1 and NQO-1, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 12 h. Total RNA was extracted and subjected to Q-PCR analysis. Relative mRNA levels were normalized with β-actin mRNA. D. To determine the protein expression levels of HO-1, NQO-1 and Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) for 24 h. Total cell lysates were prepared and subjected to western blot analysis to monitor the expression levels of HO-1, NQO-1 and Nrf2. E. To determine the Nrf2 transcriptional activity, HNDFs were transiently transfected with ARE promoter construct using lipofectamine and incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 6 h. Cell lysates were mixed with luciferase reagents and quantified using an illuminometer. Relative ARE promoter activity was calculated by dividing the relative luciferace unit (RLU) of treated cells by RLU of untreated cells (NG). F. To determine the nuclear localization of Nrf2, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence or HG (30 mM) for 2 h. The protein expression and localization of Nrf2 was measured by immunofluorescence using Nrf2 specific primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). The subcellular and nuclear localization of Nrf2 was photographed using a fluoroscence microscope. DAPI (1 μM) was used to stain the nucleus. G. HNDFs were pre-incubated with AKT, ERK1/2, JNK/SAPK and p38 MAPK inhbitors LY294002 (LY, 30 μM), PD98059 (PD, 30 μM), SP600125 (SP, 30 μM) and SB203580 (SB, 30 μM), respectively for 2 h and then incubated with ANM (10 μM) in the presence of HG (30 mM) 2 h. Cytoplasmic and nuclear fractions were prepared and subjected to western blot analysis. GAPDH and histone H3 served as internal controls for the cytoplasmic and nuclear fraction, respectively. H. The Keap-1 protein expression level was determined by western blotting. I. Effect of ANM on ubiquitination of Keap-1. Equivalent amount of proteins were immune-precipitated with Keap-1 antibody and visualized by western blotting with ubiquitin antibody. Histogram shows the percentage of ubiquinated Keap-1. Results expressed as mean ± S.E.M of three indipendent expriments. Statistical significance was set at Ф P

    Article Snippet: Antibodies against cyclin D1, cyclin B1, cyclin E, CDK2, CDK4, CDK6, Cdc2, phos-pRb, p16INK4A , p21CIP1 , acety-p52, phos-p53, phos-FoxO1, FoxO1, phos-JNK/SAPK, JNK/SAPK, phos-p38 MAPK, p38 MAPK, phos-ERK1/2, ERK1/2, Phos-AKT, AKT, histone H3, phos-SIRT-1, SIRT-1, SIRT-3, SIRT-6 and Keap-1 were obtained from Cell Signaling Technology, Danvers, MA.

    Techniques: DPPH Assay, Expressing, Incubation, Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Construct, Luciferase, Immunofluorescence, Microscopy, Staining

    Antcin M inhibits HG-induced senescence in HNDFs A. ANM inhibits HG-induced intracellular ROS. HNDFs were incubated with ANM (10 μM) or NAC (100 μM) either in the presence or absence of HG (30 mM) for 24 h. The intracellular ROS level was determined by flow cytometry using DCH-DA flurogenic probe. The left panel shows representative figures and the right panel shows quantitative analysis of intracellular ROS in HG-treated HNDFs. B. ANM eliminates HG-induced senescenece. HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 72 h. Cellular senescence was determined by SA-β-gal assay. The left panel shows representative figures and right panel shows quantitative analysis of SA-β-gal positive cells per microscopic field. C. ANM provides SMP30 expression. HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 72 h. The protein expression of SMP30 was measured by immunofluroscence using SMP30 specific primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). The cellular localization of SMP30 was photographed using a fluroscence microscope. DAPI (1 μM) was used to stain the nucleus. D. - G. To determine the effect of ANM on senescence-associated protein expression, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 72 h. Immunoblotting analysis was used to determine the protein levels with corresponding specific antibodies. H. p38 MAPK, JNK/SAPK and AKT triggers HG-induced senescence. HNDFs were exposed to HG (30 mM) in the presence or absence of p38 MAPK, JNK/SAPK, ERK1/2 and AKT inhibitors SB203580 (SB, 30 μM), SP600125 (SP, 30 μM), PD98059 (PD, 30 μM) and LY294002 (LY, 30 μM) for 72 h. Cellular senescence was determined by SA-β-gal activity assay. Results expressed as mean ± S.E.M of three indipendent expriments. Statistical significance at Ф , P

    Journal: Oncotarget

    Article Title: A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1

    doi: 10.18632/oncotarget.11229

    Figure Lengend Snippet: Antcin M inhibits HG-induced senescence in HNDFs A. ANM inhibits HG-induced intracellular ROS. HNDFs were incubated with ANM (10 μM) or NAC (100 μM) either in the presence or absence of HG (30 mM) for 24 h. The intracellular ROS level was determined by flow cytometry using DCH-DA flurogenic probe. The left panel shows representative figures and the right panel shows quantitative analysis of intracellular ROS in HG-treated HNDFs. B. ANM eliminates HG-induced senescenece. HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 72 h. Cellular senescence was determined by SA-β-gal assay. The left panel shows representative figures and right panel shows quantitative analysis of SA-β-gal positive cells per microscopic field. C. ANM provides SMP30 expression. HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 72 h. The protein expression of SMP30 was measured by immunofluroscence using SMP30 specific primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). The cellular localization of SMP30 was photographed using a fluroscence microscope. DAPI (1 μM) was used to stain the nucleus. D. - G. To determine the effect of ANM on senescence-associated protein expression, HNDFs were incubated with ANM (10 μM) or NAC (100 μM) in the presence or absence of HG (30 mM) for 72 h. Immunoblotting analysis was used to determine the protein levels with corresponding specific antibodies. H. p38 MAPK, JNK/SAPK and AKT triggers HG-induced senescence. HNDFs were exposed to HG (30 mM) in the presence or absence of p38 MAPK, JNK/SAPK, ERK1/2 and AKT inhibitors SB203580 (SB, 30 μM), SP600125 (SP, 30 μM), PD98059 (PD, 30 μM) and LY294002 (LY, 30 μM) for 72 h. Cellular senescence was determined by SA-β-gal activity assay. Results expressed as mean ± S.E.M of three indipendent expriments. Statistical significance at Ф , P

    Article Snippet: Antibodies against cyclin D1, cyclin B1, cyclin E, CDK2, CDK4, CDK6, Cdc2, phos-pRb, p16INK4A , p21CIP1 , acety-p52, phos-p53, phos-FoxO1, FoxO1, phos-JNK/SAPK, JNK/SAPK, phos-p38 MAPK, p38 MAPK, phos-ERK1/2, ERK1/2, Phos-AKT, AKT, histone H3, phos-SIRT-1, SIRT-1, SIRT-3, SIRT-6 and Keap-1 were obtained from Cell Signaling Technology, Danvers, MA.

    Techniques: Incubation, Flow Cytometry, Cytometry, β-Gal Assay, Expressing, Microscopy, Staining, β-Gal Activity Assay