phospho mek1  (Cell Signaling Technology Inc)

 
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    Name:
    MEK1 Antibody
    Description:
    MEK1 and MEK2 also called MAPK or Erk kinases are dual specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation 1 3 Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221 located in the activation loop of subdomain VIII by Raf like molecules MEK1 2 is activated by a wide variety of growth factors and cytokines and also by membrane depolarization and calcium influx 1 4 Constitutively active forms of MEK1 2 are sufficient for the transformation of NIH 3T3 cells or the differentiation of PC 12 cells 4 MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII
    Catalog Number:
    9124
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to human MEK1. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc phospho mek1
    A : NES-B3T cells have higher levels of <t>MEK1</t> phosphorylation at an inhibitory site than NES-G2T cells. Total MEK1/2 and β-tubulin were used as loading controls. B : acid exposure increases MEK1/2 activity (measured as ERK1/2 phosphorylation) in
    MEK1 and MEK2 also called MAPK or Erk kinases are dual specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation 1 3 Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221 located in the activation loop of subdomain VIII by Raf like molecules MEK1 2 is activated by a wide variety of growth factors and cytokines and also by membrane depolarization and calcium influx 1 4 Constitutively active forms of MEK1 2 are sufficient for the transformation of NIH 3T3 cells or the differentiation of PC 12 cells 4 MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII
    https://www.bioz.com/result/phospho mek1/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho mek1 - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus"

    Article Title: Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.90262.2008

    A : NES-B3T cells have higher levels of MEK1 phosphorylation at an inhibitory site than NES-G2T cells. Total MEK1/2 and β-tubulin were used as loading controls. B : acid exposure increases MEK1/2 activity (measured as ERK1/2 phosphorylation) in
    Figure Legend Snippet: A : NES-B3T cells have higher levels of MEK1 phosphorylation at an inhibitory site than NES-G2T cells. Total MEK1/2 and β-tubulin were used as loading controls. B : acid exposure increases MEK1/2 activity (measured as ERK1/2 phosphorylation) in

    Techniques Used: Activity Assay

    Acid increases MEK1/2 phosphorylation at activating sites (serines 217/221) in both cell lines, but acid increases ERK1/2 phosphorylation only in NES-G2T cells. Total ERK1/2, total MEK1/2, and β-tubulin were used as loading controls.
    Figure Legend Snippet: Acid increases MEK1/2 phosphorylation at activating sites (serines 217/221) in both cell lines, but acid increases ERK1/2 phosphorylation only in NES-G2T cells. Total ERK1/2, total MEK1/2, and β-tubulin were used as loading controls.

    Techniques Used:

    Phosphorylation of MEK1 on threonine 286 in esophageal squamous epithelium from 2 representative patients with GERD with Barrett's esophagus and 2 representative patients with GERD without Barrett's esophagus. Note expression of the inhibitory form of
    Figure Legend Snippet: Phosphorylation of MEK1 on threonine 286 in esophageal squamous epithelium from 2 representative patients with GERD with Barrett's esophagus and 2 representative patients with GERD without Barrett's esophagus. Note expression of the inhibitory form of

    Techniques Used: Expressing

    Acid perfusion induces MEK1/2 phosphorylation on serines 217/222 in esophageal squamous epithelium of patients with GERD with and without Barrett's esophagus in vivo. A : representative Western blot demonstrating MEK1/2 phosphorylation and total MEK1/2
    Figure Legend Snippet: Acid perfusion induces MEK1/2 phosphorylation on serines 217/222 in esophageal squamous epithelium of patients with GERD with and without Barrett's esophagus in vivo. A : representative Western blot demonstrating MEK1/2 phosphorylation and total MEK1/2

    Techniques Used: In Vivo, Western Blot

    2) Product Images from "Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †"

    Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01282-09

    Mechanisms of inhibition of IL-2 expression by VIP. (A) Human T cells were cultured with medium (unstim) or CD3/CD28 activated without or with VIP (10 −7 M). After 4 h, nuclear proteins were isolated and assayed for DNA binding to NF-κB, AP-1, or NFAT consensus sites by EMSA and expressed as densitometric units relative to the binding of the constitutive nuclear factor NF-Y. The error bars indicate SD. (B) After 3 h, whole-cell extracts were analyzed by Western blotting for c-Jun and phospho-c-Jun expression. NFATp levels were determined in both nuclear and cytoplasmic extracts and expressed as densitometric units relative to laminin B or β-tubulin. (C) After 1 h of culture, cell lysates were prepared, and activation of ERK1/2 and MEK1 was determined by Western blotting with phosphospecific Abs and expressed as densitometric units normalized for total ERK1/2 or MEK1. The kinase activity of Raf1 was determined by assaying the phosphotransferase activity toward GST-MEK1. (D) Activation of Rap1 and Ras was determined as described in Materials and Methods. The inactive forms of Raf1 were measured in cell lysates by Western blotting for phospho-Ser 43 -Raf1 and phospho-Ser 259 -Raf1. n = 3 or 4. *, P
    Figure Legend Snippet: Mechanisms of inhibition of IL-2 expression by VIP. (A) Human T cells were cultured with medium (unstim) or CD3/CD28 activated without or with VIP (10 −7 M). After 4 h, nuclear proteins were isolated and assayed for DNA binding to NF-κB, AP-1, or NFAT consensus sites by EMSA and expressed as densitometric units relative to the binding of the constitutive nuclear factor NF-Y. The error bars indicate SD. (B) After 3 h, whole-cell extracts were analyzed by Western blotting for c-Jun and phospho-c-Jun expression. NFATp levels were determined in both nuclear and cytoplasmic extracts and expressed as densitometric units relative to laminin B or β-tubulin. (C) After 1 h of culture, cell lysates were prepared, and activation of ERK1/2 and MEK1 was determined by Western blotting with phosphospecific Abs and expressed as densitometric units normalized for total ERK1/2 or MEK1. The kinase activity of Raf1 was determined by assaying the phosphotransferase activity toward GST-MEK1. (D) Activation of Rap1 and Ras was determined as described in Materials and Methods. The inactive forms of Raf1 were measured in cell lysates by Western blotting for phospho-Ser 43 -Raf1 and phospho-Ser 259 -Raf1. n = 3 or 4. *, P

    Techniques Used: Inhibition, Expressing, Cell Culture, Isolation, Binding Assay, Western Blot, Activation Assay, Activity Assay

    3) Product Images from "Prompt meningeal reconstruction mediated by oxygen-sensitive AKAP12 scaffolding protein after central nervous system injury"

    Article Title: Prompt meningeal reconstruction mediated by oxygen-sensitive AKAP12 scaffolding protein after central nervous system injury

    Journal: Nature communications

    doi: 10.1038/ncomms5952

    Schematic model for prompt meningeal reconstruction and its molecular mechanism ( a ) Under normal conditions, meningeal cells maintain a high level of AKAP12 because of the high concentrations of TGF-β1, RA and oxygen. AKAP12 maintains the epithelial properties of the meninges by inhibiting the TGF-β1 pathway. ( b ) Immediately after injury, hypoxia due to vessel damage de-represses the TGF-β1-SNAI1 pathway by suppressing the expression of AKAP12 resulting in meningeal cells' EMT. Meningeal cells transited to the mesenchymal state by AKAP12 suppression invade into the site of the lesion. ( c ) At a later stage of repair, neo-microvessels formed by angiogenesis resupply oxygen to the site of the lesion. AKAP12 recovered by reoxygenation represses the TGF-β1-SNAI1 pathway, resulting in re-epithelialization of meningeal cells. ( d ) The diagram for the molecular mechanism of prompt meningeal reconstruction. Under co-treatment with RA and TGF-β1, the Smad pathway is directly blocked by RA, and non-Smad pathways are indirectly inhibited by high levels of AKAP12, the expression of which is induced by both RA and TGF-β1 under normoxic condition. In the hypoxic condition, AKAP12 expression is suppressed through RARβ repression. Then, the suppression of AKAP12 levels increases the level of SNAI1 by de-repression of the PKC/c-Raf/MEK1 and PKC/P38 pathways of TGF-β1, resulting in EMT.
    Figure Legend Snippet: Schematic model for prompt meningeal reconstruction and its molecular mechanism ( a ) Under normal conditions, meningeal cells maintain a high level of AKAP12 because of the high concentrations of TGF-β1, RA and oxygen. AKAP12 maintains the epithelial properties of the meninges by inhibiting the TGF-β1 pathway. ( b ) Immediately after injury, hypoxia due to vessel damage de-represses the TGF-β1-SNAI1 pathway by suppressing the expression of AKAP12 resulting in meningeal cells' EMT. Meningeal cells transited to the mesenchymal state by AKAP12 suppression invade into the site of the lesion. ( c ) At a later stage of repair, neo-microvessels formed by angiogenesis resupply oxygen to the site of the lesion. AKAP12 recovered by reoxygenation represses the TGF-β1-SNAI1 pathway, resulting in re-epithelialization of meningeal cells. ( d ) The diagram for the molecular mechanism of prompt meningeal reconstruction. Under co-treatment with RA and TGF-β1, the Smad pathway is directly blocked by RA, and non-Smad pathways are indirectly inhibited by high levels of AKAP12, the expression of which is induced by both RA and TGF-β1 under normoxic condition. In the hypoxic condition, AKAP12 expression is suppressed through RARβ repression. Then, the suppression of AKAP12 levels increases the level of SNAI1 by de-repression of the PKC/c-Raf/MEK1 and PKC/P38 pathways of TGF-β1, resulting in EMT.

    Techniques Used: Expressing

    AKAP12 regulates SNAI1 expression through the non-Smad pathways of TGF-β1 ( a ) AKAP12-knockdown cells were serum starved for 6 h and then treated with TGF-β1 (10 ng ml −1 ) and/or RA (10 μM) for 20 min and 24 h. Inhibition of AKAP12 activated the PKC/C-raf/MEK1 and p38 pathways among the examined non-Smad pathways of TGF-β1. Activation of these pathways was closely correlated with SNAI1 expression. This data represent the results from independent three experiments. R, RA; T, TGF-β1; TR, co-treatment of TGF-β1 and RA; C, Cont; A, AKAP12. ( b ) Specific inhibitors for PKC, C-raf, MEK1 and p38 blocked the upregulation of SNAI1 induced by AKAP12 knockdown. After serum starvation for 24 h, inhibitors were added to AKAP12-knockdown ARPE-19 cells treated with TGF-β1 (10 ng ml −1 ) and RA (10 μM) for 24 h (mean ± s.d., n = 3, analysis of variance (ANOVA) followed by Tukey-Kramer test: * P
    Figure Legend Snippet: AKAP12 regulates SNAI1 expression through the non-Smad pathways of TGF-β1 ( a ) AKAP12-knockdown cells were serum starved for 6 h and then treated with TGF-β1 (10 ng ml −1 ) and/or RA (10 μM) for 20 min and 24 h. Inhibition of AKAP12 activated the PKC/C-raf/MEK1 and p38 pathways among the examined non-Smad pathways of TGF-β1. Activation of these pathways was closely correlated with SNAI1 expression. This data represent the results from independent three experiments. R, RA; T, TGF-β1; TR, co-treatment of TGF-β1 and RA; C, Cont; A, AKAP12. ( b ) Specific inhibitors for PKC, C-raf, MEK1 and p38 blocked the upregulation of SNAI1 induced by AKAP12 knockdown. After serum starvation for 24 h, inhibitors were added to AKAP12-knockdown ARPE-19 cells treated with TGF-β1 (10 ng ml −1 ) and RA (10 μM) for 24 h (mean ± s.d., n = 3, analysis of variance (ANOVA) followed by Tukey-Kramer test: * P

    Techniques Used: Expressing, Inhibition, Activation Assay

    4) Product Images from "PV1 Is a Key Structural Component for the Formation of the Stomatal and Fenestral Diaphragms"

    Article Title: PV1 Is a Key Structural Component for the Formation of the Stomatal and Fenestral Diaphragms

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-08-0593

    Erk1/2 MAP kinase activity is required for the upregulation of PV1 as well as for the morphological changes induced by PMA in ECs. PMA activates the Erk1/2 MAP kinase pathway in ECs as demonstrated by immunoblotting with phospho-specific antibodies to components of the pathway such as MEK1, Erk1/2, and Elk-1, a downstream effector of Erk1/2 (F). MEK1, Erk1/2, and β-actin (to show the loading, Actin) were probed on the same membrane. (G) Inhibitors of MEK1 such as U0126 and PD98059 prevent the upregulation of PV1 by PMA in a dose-dependent manner, as shown by immunoblotting with anti-PV1C pAb. U0124, an inactive analog of U0126 is without effect. Immunoblotting with anti β-actin mAb shows the loading (Actin). Same inhibitors of MEK1, U0126 (D), and PD98059 (E), prevent the phenotype change of ECs induced by PMA (A), whereas U0124 has no effect (C). Control ECs (A) treated with 50 nM PMA or (B) without treatment.
    Figure Legend Snippet: Erk1/2 MAP kinase activity is required for the upregulation of PV1 as well as for the morphological changes induced by PMA in ECs. PMA activates the Erk1/2 MAP kinase pathway in ECs as demonstrated by immunoblotting with phospho-specific antibodies to components of the pathway such as MEK1, Erk1/2, and Elk-1, a downstream effector of Erk1/2 (F). MEK1, Erk1/2, and β-actin (to show the loading, Actin) were probed on the same membrane. (G) Inhibitors of MEK1 such as U0126 and PD98059 prevent the upregulation of PV1 by PMA in a dose-dependent manner, as shown by immunoblotting with anti-PV1C pAb. U0124, an inactive analog of U0126 is without effect. Immunoblotting with anti β-actin mAb shows the loading (Actin). Same inhibitors of MEK1, U0126 (D), and PD98059 (E), prevent the phenotype change of ECs induced by PMA (A), whereas U0124 has no effect (C). Control ECs (A) treated with 50 nM PMA or (B) without treatment.

    Techniques Used: Activity Assay

    5) Product Images from "Regulation of ?2B-Adrenergic Receptor-mediated Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Activation by ADP-ribosylation Factor 1 *"

    Article Title: Regulation of ?2B-Adrenergic Receptor-mediated Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Activation by ADP-ribosylation Factor 1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.267286

    Activation of MEK1 and Raf1 by the active form of ARF1. A , activation of MEK1 by ARF1Q71L. pMEK1 , phosphorylated MEK1; Ctrl , control. B , activation of Raf1 by ARF1Q71L. pRaf1 , phosphorylated Raf1. C , expression of the dominant-negative mutant Raf1K375M,
    Figure Legend Snippet: Activation of MEK1 and Raf1 by the active form of ARF1. A , activation of MEK1 by ARF1Q71L. pMEK1 , phosphorylated MEK1; Ctrl , control. B , activation of Raf1 by ARF1Q71L. pRaf1 , phosphorylated Raf1. C , expression of the dominant-negative mutant Raf1K375M,

    Techniques Used: Activation Assay, Expressing, Dominant Negative Mutation

    6) Product Images from "PARTICIPATION OF VITAMIN D-UPREGULATED PROTEIN 1 (TXNIP)-ASK1-JNK1 SIGNALOSOME IN THE ENHANCEMENT OF AML CELL DEATH BY A POST-CYTOTOXIC DIFFERENTIATION REGIMEN."

    Article Title: PARTICIPATION OF VITAMIN D-UPREGULATED PROTEIN 1 (TXNIP)-ASK1-JNK1 SIGNALOSOME IN THE ENHANCEMENT OF AML CELL DEATH BY A POST-CYTOTOXIC DIFFERENTIATION REGIMEN.

    Journal: The Journal of steroid biochemistry and molecular biology

    doi: 10.1016/j.jsbmb.2018.11.015

    In vitro assays of ASK1 kinase activity showing increased enzyme activity in ECD using two different substrates (note the fourth lane in A and B) to demonstrate consistency. HL60 cell lysates (100 μg) were immunoprecipitated using ASK1 cross-linked column, then incubated with 1 μg of recombinant human p38 or MEK1 proteins as substrates for ASK1-immunoprecipitated kinase assay in the presence of ATP. Western blots were run and probed for Phospho-p38 (Thr180/Tyr182) or for Total-p38, and Phospho-MEK1 (Ser217/221) or Total-MEK1.
    Figure Legend Snippet: In vitro assays of ASK1 kinase activity showing increased enzyme activity in ECD using two different substrates (note the fourth lane in A and B) to demonstrate consistency. HL60 cell lysates (100 μg) were immunoprecipitated using ASK1 cross-linked column, then incubated with 1 μg of recombinant human p38 or MEK1 proteins as substrates for ASK1-immunoprecipitated kinase assay in the presence of ATP. Western blots were run and probed for Phospho-p38 (Thr180/Tyr182) or for Total-p38, and Phospho-MEK1 (Ser217/221) or Total-MEK1.

    Techniques Used: In Vitro, Activity Assay, Immunoprecipitation, Incubation, Recombinant, Kinase Assay, Western Blot

    7) Product Images from "MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization"

    Article Title: MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7972

    MEK1 maintains liver CSC self-renewal dependent on SIRT1 Non-CSCs (Huh7-Nanog Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM U0126 or DMSO for 48 hours. Liver CSCs (Huh7-Nanog Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs ( C ) and non-CSCs ( A ) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs ( D ) and non-CSCs ( B ) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. ( E – F ) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = 0.52 × (W) 2 × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.
    Figure Legend Snippet: MEK1 maintains liver CSC self-renewal dependent on SIRT1 Non-CSCs (Huh7-Nanog Neg cells) were prepared with overexpression SIRT1, and grown with 5 μM U0126 or DMSO for 48 hours. Liver CSCs (Huh7-Nanog Pos cells) co-cultured with 5 μM U0126 for 24 hours previously and overexpression SIRT1 for next 24 hours. Colony analysis of CSCs ( C ) and non-CSCs ( A ) which were cultured for 14 days and stained with crystal violet. Sphere analysis of CSCs ( D ) and non-CSCs ( B ) which were cultured for 7 days in non-adhesive culture system. All counting were performed in triplicate. ( E – F ) CSCs, MEK1 inhibition CSCs and SIRT1 overexpression while MEK1 inhibition CSCs were prepared for 14 days, then subcutaneous injected in NOD-SCID mice (CSCs control group 4 mice, other two groups 8 mice each). Tumor sizes were measured with calipers in three dimensions every other day. Tumor volumes were calculated using the formula: tumor volume (cm 3 ) = 0.52 × (W) 2 × (L), where L is length and W is width. We counted and weight the tumors, 30 days later.

    Techniques Used: Over Expression, Cell Culture, Staining, Inhibition, Injection, Mouse Assay

    Relationship of p-MEK1/SIRT1 and HCC clinical prognosis ( A ) p-MEK1, SIRT1 and Nanog protein expression were detected by IHC analysis in 148 HCC patients, representative images were shown. ( B ) Analysis correlation between p-MEK1 and SIRT1 expression in 148 HCC patients with Person chi-square test. Correlation analysis of p-MEK1/SIRT1 and Nanog expression in same tissue samples. * P
    Figure Legend Snippet: Relationship of p-MEK1/SIRT1 and HCC clinical prognosis ( A ) p-MEK1, SIRT1 and Nanog protein expression were detected by IHC analysis in 148 HCC patients, representative images were shown. ( B ) Analysis correlation between p-MEK1 and SIRT1 expression in 148 HCC patients with Person chi-square test. Correlation analysis of p-MEK1/SIRT1 and Nanog expression in same tissue samples. * P

    Techniques Used: Expressing, Immunohistochemistry

    MEK1 signaling activity is required for the maintenance of liver CSC self-renewal ( A ) Huh7-Nanog Pos cells were co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) in sphere-forming conditions for 7 days, counted at the same magnification. ( B ) Huh7-Nanog Pos cells were treated with different concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and grown for 14 days. Cells were stained with crystal violet and counted. ( C ) Western blot analysis of stemness protein expression in Huh7- and PLC/PRF/5-Nanog Pos cells, which co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. ( D ) Huh7-Nanog Pos cells were treated with 5 μM U0126 for 14 days, while the negative control treated with DMSO for 14 days. Then we subcutaneous injected 1 × 10 2 , 1 × 10 3 , 1 × 10 4 cells into NOD-SCID mice. After 30 days, we harvested and counted the tumors. Extreme Limiting Dilution Analysis was acquired from http://bioinf.wehi.edu.au/software/elda/ .
    Figure Legend Snippet: MEK1 signaling activity is required for the maintenance of liver CSC self-renewal ( A ) Huh7-Nanog Pos cells were co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) in sphere-forming conditions for 7 days, counted at the same magnification. ( B ) Huh7-Nanog Pos cells were treated with different concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and grown for 14 days. Cells were stained with crystal violet and counted. ( C ) Western blot analysis of stemness protein expression in Huh7- and PLC/PRF/5-Nanog Pos cells, which co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. ( D ) Huh7-Nanog Pos cells were treated with 5 μM U0126 for 14 days, while the negative control treated with DMSO for 14 days. Then we subcutaneous injected 1 × 10 2 , 1 × 10 3 , 1 × 10 4 cells into NOD-SCID mice. After 30 days, we harvested and counted the tumors. Extreme Limiting Dilution Analysis was acquired from http://bioinf.wehi.edu.au/software/elda/ .

    Techniques Used: Activity Assay, Cell Culture, Staining, Western Blot, Expressing, Planar Chromatography, Negative Control, Injection, Mouse Assay, Software

    MEK1 knockdown suppresses liver CSC self-renewal and tumorgenetic capacity ( A ) Western blot analysis MEK1 and the substrate ERK1/2 expression in Huh7- and PLC/PRF/5-Nanog Pos cells which depleted MEK1 with two individual lentiviruses for 48 hours. ( B ) Effect of MEK1 knockdown on cellular growth rates of Huh7- and PLC/PRF/5- Nanog Pos cells. CCK8 assay was performed after transfection with indicated times. Cell lysates were obtained from cells transiently transfected with either MEK1 shRNA or negative control shRNA. ( C ) Huh7- and PLC/PRF/5- Nanog Pos cells which transfected with MEK1 shRNA or negative control shRNA cultured under non-adhesive culture system for 7 days. ( D ) Huh7- and PLC/PRF/5-Nanog Pos cells were transduced with lentiviruses expressing the indicated shRNA. Cells were grown for 14 days and stained with crystal violet. ( E ) Western blot analysis of stemness-related proteins in MEK1-depleted cells, relative to control.
    Figure Legend Snippet: MEK1 knockdown suppresses liver CSC self-renewal and tumorgenetic capacity ( A ) Western blot analysis MEK1 and the substrate ERK1/2 expression in Huh7- and PLC/PRF/5-Nanog Pos cells which depleted MEK1 with two individual lentiviruses for 48 hours. ( B ) Effect of MEK1 knockdown on cellular growth rates of Huh7- and PLC/PRF/5- Nanog Pos cells. CCK8 assay was performed after transfection with indicated times. Cell lysates were obtained from cells transiently transfected with either MEK1 shRNA or negative control shRNA. ( C ) Huh7- and PLC/PRF/5- Nanog Pos cells which transfected with MEK1 shRNA or negative control shRNA cultured under non-adhesive culture system for 7 days. ( D ) Huh7- and PLC/PRF/5-Nanog Pos cells were transduced with lentiviruses expressing the indicated shRNA. Cells were grown for 14 days and stained with crystal violet. ( E ) Western blot analysis of stemness-related proteins in MEK1-depleted cells, relative to control.

    Techniques Used: Western Blot, Expressing, Planar Chromatography, CCK-8 Assay, Transfection, shRNA, Negative Control, Cell Culture, Transduction, Staining

    MEK1 mainly promotes SIRT1 expression in HCC population ( A ) Western blot analysis the sirtuins expression level in CSCs and non-CSCs, compared with U0126 inhibited CSCs. ( B ) Western blot analysis SIRT1 expression in CSCs after co-cultured with indicated concentration of U0126 or PD98059 ( D ) for 48 hours. ( C ) Western blot analysis SIRT1 expression in CSCs which cultured with 5 μM U0126 for indicated times. ( E ) Western blot analysis SIRT1 expression in CSCs transduced with lentiviruses expressing the indicated shRNA for 48 hours. Those experiments were repeated in two HCC cell lines (Huh7 and PLC/PRF/5).
    Figure Legend Snippet: MEK1 mainly promotes SIRT1 expression in HCC population ( A ) Western blot analysis the sirtuins expression level in CSCs and non-CSCs, compared with U0126 inhibited CSCs. ( B ) Western blot analysis SIRT1 expression in CSCs after co-cultured with indicated concentration of U0126 or PD98059 ( D ) for 48 hours. ( C ) Western blot analysis SIRT1 expression in CSCs which cultured with 5 μM U0126 for indicated times. ( E ) Western blot analysis SIRT1 expression in CSCs transduced with lentiviruses expressing the indicated shRNA for 48 hours. Those experiments were repeated in two HCC cell lines (Huh7 and PLC/PRF/5).

    Techniques Used: Expressing, Western Blot, Cell Culture, Concentration Assay, Transduction, shRNA, Planar Chromatography

    MEK1 keeps SIRT1 protein stability through proteasomal degradation inhibitory ( A ) We co-cultured proteasome inhibition, MEK1 inhibition or knockdown liver CSCs with CHX (10 ng/ml) for indicated times. Western blots analyzed expression of SIRT1. Grey level was measured triplicated independently. ( B ) Analysis of SIRT1 expression in liver CSCs by western blots. MEK1 deletion or inhibition (U0126, 5 μM) CSCs was cultured for 48 hours, then combined with or without Proteasome inhibitor (MG132, 10 μM) for 8 hours, before harvested. Those proteins were compared with CSCs of DMSO treatment with or without MG132. Grey level was measured and marked. ( C ) MEK1 inhibition or knockdown CSCs (Huh7- and PLC/PRF/5-Nanog Pos cells) were treated with 10 μM MG132 for 8 hours before harvest. Total protein extracts from an equivalent number of seedlings were prepared for Co-IP in same conditions and analyzed using immunoblot with the Poly-Ub antibody.
    Figure Legend Snippet: MEK1 keeps SIRT1 protein stability through proteasomal degradation inhibitory ( A ) We co-cultured proteasome inhibition, MEK1 inhibition or knockdown liver CSCs with CHX (10 ng/ml) for indicated times. Western blots analyzed expression of SIRT1. Grey level was measured triplicated independently. ( B ) Analysis of SIRT1 expression in liver CSCs by western blots. MEK1 deletion or inhibition (U0126, 5 μM) CSCs was cultured for 48 hours, then combined with or without Proteasome inhibitor (MG132, 10 μM) for 8 hours, before harvested. Those proteins were compared with CSCs of DMSO treatment with or without MG132. Grey level was measured and marked. ( C ) MEK1 inhibition or knockdown CSCs (Huh7- and PLC/PRF/5-Nanog Pos cells) were treated with 10 μM MG132 for 8 hours before harvest. Total protein extracts from an equivalent number of seedlings were prepared for Co-IP in same conditions and analyzed using immunoblot with the Poly-Ub antibody.

    Techniques Used: Cell Culture, Inhibition, Western Blot, Expressing, Planar Chromatography, Co-Immunoprecipitation Assay

    MEK1 inhibitor decreases liver CSCs proliferation ability in vitro ( A ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells under different U0126 concentrations treatment as indicated (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were seeded (1 × 10 3 ) and cultured for another 6 days before analyzed with CCK8. ( B ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells were cultured with or without 5 μM U0126 for 48 hours. Cells were harvested for immunofluorescence (IF) analysis by anti-Ki67 antibodies. Scale bar, 10 μm. ( C ) Cell cycle profiles of 5 μM U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-Nanog Pos cells followed by treatment with Sodium butyrate. Percentage in each histogram indicates the portion of cells remaining in each cell cycle phase.
    Figure Legend Snippet: MEK1 inhibitor decreases liver CSCs proliferation ability in vitro ( A ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells under different U0126 concentrations treatment as indicated (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM) were seeded (1 × 10 3 ) and cultured for another 6 days before analyzed with CCK8. ( B ) Huh7-Nanog Pos and PLC/PRF/5-Nanog Pos cells were cultured with or without 5 μM U0126 for 48 hours. Cells were harvested for immunofluorescence (IF) analysis by anti-Ki67 antibodies. Scale bar, 10 μm. ( C ) Cell cycle profiles of 5 μM U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-Nanog Pos cells followed by treatment with Sodium butyrate. Percentage in each histogram indicates the portion of cells remaining in each cell cycle phase.

    Techniques Used: In Vitro, Planar Chromatography, Cell Culture, Immunofluorescence, Negative Control

    8) Product Images from "Melittin suppresses cathepsin S-induced invasion and angiogenesis via blocking of the VEGF-A/VEGFR-2/MEK1/ERK1/2 pathway in human hepatocellular carcinoma"

    Article Title: Melittin suppresses cathepsin S-induced invasion and angiogenesis via blocking of the VEGF-A/VEGFR-2/MEK1/ERK1/2 pathway in human hepatocellular carcinoma

    Journal: Oncology Letters

    doi: 10.3892/ol.2015.3957

    Effects of melittin on the phosphorylation/activation of VEGF-A/VEGFR-2/MEK1/ERK1/2 signaling pathway. Melittin specifically decreased the expression of phosphorylated/activated Cat S, VEGF-A, p-VEGFR-2, Ras, p-Raf, p-MEK1 and p-ERK1/2 in Mock/MHCC97-H cells, however, did not affect shRNA-Cat S/MHCC97-H cells. Results are from a representative experiment performed with qualitatively similar results. β-actin served as an internal control in each sample. VEGFR, vascular endothelial growth factor receptor; MEK, mitogen-activated protein kinase; ERK, extracellular regulated mitogen-activated protein kinase; p, phosphorylated, shRNA, small hairpin RNA.
    Figure Legend Snippet: Effects of melittin on the phosphorylation/activation of VEGF-A/VEGFR-2/MEK1/ERK1/2 signaling pathway. Melittin specifically decreased the expression of phosphorylated/activated Cat S, VEGF-A, p-VEGFR-2, Ras, p-Raf, p-MEK1 and p-ERK1/2 in Mock/MHCC97-H cells, however, did not affect shRNA-Cat S/MHCC97-H cells. Results are from a representative experiment performed with qualitatively similar results. β-actin served as an internal control in each sample. VEGFR, vascular endothelial growth factor receptor; MEK, mitogen-activated protein kinase; ERK, extracellular regulated mitogen-activated protein kinase; p, phosphorylated, shRNA, small hairpin RNA.

    Techniques Used: Activation Assay, Expressing, shRNA

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    Cell Signaling Technology Inc mek1 2
    PLD1 modulates TNF-α expression and release via phosphorylation of <t>MEK1/2</t> and ERK1/2. MEFs from Pld1 +/+ and Pld1 −/− mice were stimulated with 1 mg/ml LPS for indicated time points. ( A) TNF-α release into the supernatant of MEFs after LPS stimulation was measured by ELISA. ( B – E ) Phosphorylation of SAPK/JNK, p38 and ERK1/2 was detected by Western blot after stimulation of MEFs with LPS for 30 min. and quantified via adjusted density using ImageJ. Same samples for quantification of total protein expression were used but applied to different gels/membranes. Cropped blots are shown. ( F – H ) Phosphorylation of MEK1/2 and protein abundance of EGR-1 was detected after LPS stimulation of MEFs for 30 min. using Western blot analysis and quantified via adjusted density using ImageJ. B-tubulin serves as loading control for the detection of EGR-1. Cropped blots are shown. ( I ) Egr-1 expression in MEFs was analyzed by qRT-PCR 5 hrs. after LPS stimulation. ( J ) ΔEgr-1 expression (basal-stimulated) of Pld1 +/+ and Pld1 −/− MEFs. N = 5. ( K ) Egr-1 expression in the liver of PLD1 deficient and control mice was determined by qRT-PCR. N = 9. Bar graphs depict mean values ± s.e.m. *P
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    Cell Signaling Technology Inc phosphorylated mek1
    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and <t>MEK1</t> in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p
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    Cell Signaling Technology Inc rabbit monoclonal anti phospho mek1 2
    Effects of S100A9 and S100A8/A9 on MKP-1 and MAPK phosphorylation. ( a and b ) Western blots corresponding to MKP-1, p-p38, p-Erk1/2, <t>p-MEK1/2</t> in lung lysates from mice treated with S100A9 or S100A8/A9 for 2 h and then LPS for ( a ) 4 h or ( b ) 1 h. A representative blot is shown; β-actin was used for loading control; n =5 individual lung lysates/group in two independent analyses. Numbers indicate 1: HBSS+PBS, 2: HBSS+LPS, 3: S100A9+LPS, 4: S100A8/A9+LPS.
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    Figure 2. Stimulation of MAPK signaling by oncogenic B-Raf attenuates G 2 checkpoint function in melanoma cells. ( A ) Western immunoblot showing induction of V5-V600E B-Raf and stimulation of MAPK signaling <t>(P-MEK1/2)</t> in the melanoma line RPMI8332.
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    PLD1 modulates TNF-α expression and release via phosphorylation of MEK1/2 and ERK1/2. MEFs from Pld1 +/+ and Pld1 −/− mice were stimulated with 1 mg/ml LPS for indicated time points. ( A) TNF-α release into the supernatant of MEFs after LPS stimulation was measured by ELISA. ( B – E ) Phosphorylation of SAPK/JNK, p38 and ERK1/2 was detected by Western blot after stimulation of MEFs with LPS for 30 min. and quantified via adjusted density using ImageJ. Same samples for quantification of total protein expression were used but applied to different gels/membranes. Cropped blots are shown. ( F – H ) Phosphorylation of MEK1/2 and protein abundance of EGR-1 was detected after LPS stimulation of MEFs for 30 min. using Western blot analysis and quantified via adjusted density using ImageJ. B-tubulin serves as loading control for the detection of EGR-1. Cropped blots are shown. ( I ) Egr-1 expression in MEFs was analyzed by qRT-PCR 5 hrs. after LPS stimulation. ( J ) ΔEgr-1 expression (basal-stimulated) of Pld1 +/+ and Pld1 −/− MEFs. N = 5. ( K ) Egr-1 expression in the liver of PLD1 deficient and control mice was determined by qRT-PCR. N = 9. Bar graphs depict mean values ± s.e.m. *P

    Journal: Scientific Reports

    Article Title: Phospholipase D1 regulation of TNF-alpha protects against responses to LPS

    doi: 10.1038/s41598-018-28331-y

    Figure Lengend Snippet: PLD1 modulates TNF-α expression and release via phosphorylation of MEK1/2 and ERK1/2. MEFs from Pld1 +/+ and Pld1 −/− mice were stimulated with 1 mg/ml LPS for indicated time points. ( A) TNF-α release into the supernatant of MEFs after LPS stimulation was measured by ELISA. ( B – E ) Phosphorylation of SAPK/JNK, p38 and ERK1/2 was detected by Western blot after stimulation of MEFs with LPS for 30 min. and quantified via adjusted density using ImageJ. Same samples for quantification of total protein expression were used but applied to different gels/membranes. Cropped blots are shown. ( F – H ) Phosphorylation of MEK1/2 and protein abundance of EGR-1 was detected after LPS stimulation of MEFs for 30 min. using Western blot analysis and quantified via adjusted density using ImageJ. B-tubulin serves as loading control for the detection of EGR-1. Cropped blots are shown. ( I ) Egr-1 expression in MEFs was analyzed by qRT-PCR 5 hrs. after LPS stimulation. ( J ) ΔEgr-1 expression (basal-stimulated) of Pld1 +/+ and Pld1 −/− MEFs. N = 5. ( K ) Egr-1 expression in the liver of PLD1 deficient and control mice was determined by qRT-PCR. N = 9. Bar graphs depict mean values ± s.e.m. *P

    Article Snippet: We found PLD1 to be responsible for the phosphorylation of MEK1/2 and ERK1/2 after LPS-induced TLR4 activation.

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

    Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Journal: Molecules

    Article Title: Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    doi: 10.3390/molecules21030301

    Figure Lengend Snippet: Inhibitory effect of achillolide A on H 2 O 2 -induced phosphorylation of p44/42 MAPK and MEK1 in astrocytes. Astrocytes were treated with 175 μM of H 2 O 2 for 40 min following preincubation with achillolide A or memantine for 2 h. The levels of phosphorylated and total p44/42 MAPK ( A , B ) and phosphorylated and total MEK1 ( C ) were measured by ELISA. Relative phospho MAPK levels were calculated from the ratio: phospho MAPK levels/total MAPK levels the results are means ± SEM of two experiments ( n = 4). * p

    Article Snippet: To measure the amount of total and phosphorylated MEK1 in cell lysates of astrocytes, ELISA was performed according to the manufacturer’s protocol using the PathScan total MEK1 sandwich ELISA kit (Cell Signaling Technology) and the PathScan phosoho-MEK1 (Ser217/221) sandwich ELISA kit (Cell Signaling Technology), respectively.

    Techniques: Enzyme-linked Immunosorbent Assay

    Effects of S100A9 and S100A8/A9 on MKP-1 and MAPK phosphorylation. ( a and b ) Western blots corresponding to MKP-1, p-p38, p-Erk1/2, p-MEK1/2 in lung lysates from mice treated with S100A9 or S100A8/A9 for 2 h and then LPS for ( a ) 4 h or ( b ) 1 h. A representative blot is shown; β-actin was used for loading control; n =5 individual lung lysates/group in two independent analyses. Numbers indicate 1: HBSS+PBS, 2: HBSS+LPS, 3: S100A9+LPS, 4: S100A8/A9+LPS.

    Journal: Immunology and Cell Biology

    Article Title: S100A8/A9 and S100A9 reduce acute lung injury

    doi: 10.1038/icb.2017.2

    Figure Lengend Snippet: Effects of S100A9 and S100A8/A9 on MKP-1 and MAPK phosphorylation. ( a and b ) Western blots corresponding to MKP-1, p-p38, p-Erk1/2, p-MEK1/2 in lung lysates from mice treated with S100A9 or S100A8/A9 for 2 h and then LPS for ( a ) 4 h or ( b ) 1 h. A representative blot is shown; β-actin was used for loading control; n =5 individual lung lysates/group in two independent analyses. Numbers indicate 1: HBSS+PBS, 2: HBSS+LPS, 3: S100A9+LPS, 4: S100A8/A9+LPS.

    Article Snippet: The following antibodies were used: rabbit monoclonal anti-phospho-MEK1/2 (Ser217/221 , 1:1000 v/v), anti-phospho-p38 MAPK (Thr180 /Tyr182 , 1:1000 v/v), anti-IκBα (1:1000 v/v) and NF-κB1 p105/p50 (1:1000 v/v), and rabbit polyclonal anti-MEK1/2 (1:1000 v/v), anti-phosho-Erk1/2 MAPK (Thr202 /Tyr204 , 1:1000 v/v), anti-SIRT1 (1:1000 v/v), anti-phospho-STAT3 (1:1000 v/v), anti-STAT3 (1:1000 v/v) (all from Cell Signaling Technology, Danvers, MA, USA), and anti-MKP-1 (1:500 v/v, Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Western Blot, Mouse Assay

    Figure 2. Stimulation of MAPK signaling by oncogenic B-Raf attenuates G 2 checkpoint function in melanoma cells. ( A ) Western immunoblot showing induction of V5-V600E B-Raf and stimulation of MAPK signaling (P-MEK1/2) in the melanoma line RPMI8332.

    Journal: Cell Cycle

    Article Title: A prognostic signature of G? checkpoint function in melanoma cell lines

    doi: 10.4161/cc.24067

    Figure Lengend Snippet: Figure 2. Stimulation of MAPK signaling by oncogenic B-Raf attenuates G 2 checkpoint function in melanoma cells. ( A ) Western immunoblot showing induction of V5-V600E B-Raf and stimulation of MAPK signaling (P-MEK1/2) in the melanoma line RPMI8332.

    Article Snippet: Expression of V5V600E was monitored by immunoblot analysis using antibodies against V5 (Sigma), B-Raf (Cell Signaling) and phospho-MEK1/2 (Cell Signaling).

    Techniques: Western Blot