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  • 99
    Millipore monoclonal anti map kinase activated diphosphorylated erk 1 2 antibody
    Monoclonal Anti Map Kinase Activated Diphosphorylated Erk 1 2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc phospho erk
    Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and <t>ERK</t> or phospho-MET, <t>phospho-Akt</t> and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).
    Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 6485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti phospho erk
    The selective activation of MAPK phosphorylation induced by different magnitudes of tensile strain. Western blot of MAPK was shown as <t>p-ERK</t> and ERK ( a ), p-p38 and p38 ( b ), and <t>p-JNK</t> and JNK ( c ). d Quantification graph for the protein levels of the phosphorylated
    Anti Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology phospho extracellular signal regulated kinase
    The key signaling pathways regulating proliferation and metastasis are suppressed during Rhein treatment. Notes: ( A ) 30 or 60 μM Rhein inhibited the expression of p-ERK, p-Akt, MMP9 and CCND1 in A498 and 786-O cells at 48 h. ( B ) The quantitative results of the key molecules changing showed in ( A ) by ImageJ software. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; p-JNK, <t>phospho-c-Jun</t> N-terminal <t>kinase;</t> p-ERK, <t>phospho-extracellular</t> <t>signal-regulated</t> kinase.
    Phospho Extracellular Signal Regulated Kinase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti phospho erk
    Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The <t>4G10</t> (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho <t>ERK</t> and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.
    Anti Phospho Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho erk thr202 tyr204
    Incubation of adipose tissue with dexamethasone (0.3 µM) for 24 h or dexamethasone for 24 h together with the CNR1-specific antagonist AM281 (3 µM) for the final 4 h of incubation had no effect on the total protein levels or on the phosphorylation of HSL Ser563 a and <t>ERK</t> <t>Thr202/Tyr204</t> b ( n = 5). Data are means of densitometry analyses of p-HSL and p-ERK and normalized to the respective total protein levels ( n = 5). GAPDH was used as a loading control protein
    Phospho Erk Thr202 Tyr204, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti phospho erk
    DC-ASGPR ligation induces IL-10 from DCs dependent on <t>ERK/p38</t> contributing to the generation of antigen-specific IL-10–producing CD4 + T cells. (A) IFNDCs (10 5 ) were incubated with indicated inhibitors for 1 h, washed, and loaded with 1 µg/ml IgG4-PSA, α-LOX-1-PSA, or α-DC-ASGPR-PSA. After 24 h, culture supernatants were harvested and IL-10 production was assessed. Error bars indicate the mean ± SEM of two independent experiments with triplicate assay. (B) After 15 min of loading IFNDCs with 1 µg/ml recombinant proteins, α-DC-ASGPR-PSA, or α-LOX-1-PSA, cells were stained with indicated antibodies. Representative data from four independent experiments are presented. (C) 5 × 10 3 IFNDCs were treated with 2.5 µM PD0325901 (MEK inhibitor) or SB203580 (p38 inhibitor) for 1 h and washed thoroughly. DCs were loaded with 1 µg/ml α-DC-ASGPR-PSA or α-LOX-1-PSA. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d. CD4 + T cells were then restimulated with 1 µM PSA 30-44 for 48 h. IL-10, IFN-γ, and IL-2 in culture supernatants were assessed. Each dot represents the data generated with a single experiment. Background values acquired with control peptide (PSA 82-96 ) were substracted. (D) Purified naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with α-DC-ASGPR-PSA–loaded IFNDCs in the presence of control IgG or α–IL-10/IL-10R antibodies for 7 d. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from four independent experiments are presented on the right. (E) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml α-LOX-1-PSA in the presence or absence of 20 pg/ml IL-10. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from five independent experiments are presented in right panel. P-values were calculated with Student’s t test.
    Rabbit Anti Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 481 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology phosphorylated erk
    Effect of the calmodulin-dependent PDE inhibitor CGS and CAMKK inhibitor STO on <t>ERK</t> activation. Relative density of <t>pERK/ERK</t> measured by densitometry at 24 h in Saos-2 (A, C) or MG-63 (B, D) cells treated alone with 5 μM CdCl 2 , 5 μM CGS, 5 μM STO, 2.5 μM KN-93 or with CdCl 2 and inhibitor in co-treatment. Controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. Representative Western blots of pERK and ERK for Saos-2 (E, G) and MG-63 (F, H). Each bar represents the mean ± SEM of at least 3 independent experiments. * denotes significant from control p
    Phosphorylated Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc anti phosphoerk
    Modulation of T Cell Activation by DNA-CARζ Length and Sequence . (B) Dose-response curves for DNA ligands of varying hybridization lengths. The total ligand DNA length remained constant by adding non-hybridizing Ts. Mean ± SD (n = 3). (C) Stepwise conversion of A/T to G/C base pairs increases the potency of the 11-mer DNA ligand for inducing <t>phosphoERK</t> signaling. Scale bar, 100 μm. Mean ± SD of each ligand density measured in triplicate from one representative experiment. (D) Single ligand-receptor lifetimes by TIRF microscopy. An example distribution of lifetimes from one DNA ligand. The observed (τ obs ) and photobleach-corrected (τ corr .
    Anti Phosphoerk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).

    Journal: Nature Communications

    Article Title: A cysteine selenosulfide redox switch for protein chemical synthesis

    doi: 10.1038/s41467-020-16359-6

    Figure Lengend Snippet: Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).

    Article Snippet: Cell lysates were then analyzed by western blot using specific total MET (#37-0100 Invitrogen), total ERK2 (#SC-154 Tebu-bio), phospho-MET (Y1234/1235, clone CD26, #3077 Cell Signaling), phospho-Akt (S473, clone CD9E, #4060 Cell Signaling), phospho-ERK (T202/Y204, clone E10, #9106 Cell Signaling).

    Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Incubation, Standard Deviation, Phosphorylation Assay, Western Blot, Stripping Membranes, Scattering Assay, Isolation, Staining

    Resistance to vemurafenib is associated with single-cell variability in phosphorylated ERK levels 24 hours after treatment but not prior to treatment. A . We used Rewind to quantify dual-phospho ERK (p44/p42, pERK) levels in primed cells before and 24 hours after vemurafenib treatment. To quantify pERK levels over time, we plated two Carbon Copies and fixed one 24 hours after vemurafenib treatment and the other prior to treatment. As before, we used barcode RNA FISH probes to identify primed cells in both Carbon Copies then measured single-cell levels of total ERK and pERK by immunofluorescence. We additionally imaged multiple randomly selected positions in each well to quantify total ERK and pERK in non-primed cells. B . Barcode RNA FISH and ERK immunofluorescence images of primed cells identified in Carbon Copies fixed before vemurafenib treatment (left) and 24 hours after treatment (right). C . Quantification of average pERK immunofluorescence intensity in primed cells and non-primed cells. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. These data correspond to 1 of 2 biological replicates (See Supp. Fig. 8 for additional replicate).

    Journal: bioRxiv

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance

    doi: 10.1101/2020.03.18.996660

    Figure Lengend Snippet: Resistance to vemurafenib is associated with single-cell variability in phosphorylated ERK levels 24 hours after treatment but not prior to treatment. A . We used Rewind to quantify dual-phospho ERK (p44/p42, pERK) levels in primed cells before and 24 hours after vemurafenib treatment. To quantify pERK levels over time, we plated two Carbon Copies and fixed one 24 hours after vemurafenib treatment and the other prior to treatment. As before, we used barcode RNA FISH probes to identify primed cells in both Carbon Copies then measured single-cell levels of total ERK and pERK by immunofluorescence. We additionally imaged multiple randomly selected positions in each well to quantify total ERK and pERK in non-primed cells. B . Barcode RNA FISH and ERK immunofluorescence images of primed cells identified in Carbon Copies fixed before vemurafenib treatment (left) and 24 hours after treatment (right). C . Quantification of average pERK immunofluorescence intensity in primed cells and non-primed cells. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. These data correspond to 1 of 2 biological replicates (See Supp. Fig. 8 for additional replicate).

    Article Snippet: Immunofluorescence We performed immunofluorescence using primary antibodies targeting total ERK (L34F12 Cell Signalling #4696) and phosphorylated ERK (p44/p42 ERK D12.14.4E Cell Signalling #4370).

    Techniques: Fluorescence In Situ Hybridization, Immunofluorescence

    Synergistic effect of Grb2 and ITGA1 inhibition through ERK signaling. A. Hela cells and Jurkat cells were treated with Grb2 siRNA in the absence or presence of ITGA1 siRNA. Western blot analysis for expression of phosphorylated and total ERK, cleaved caspase-3 and cleaved PARP. B. The percentages of the sub-G0/G1 population were determined by flow cytometry in Hela cells and Jurkat cells. Data are represented as mean ± S.E.M. (n=3). *** P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Synergistic anticancer effect of Grb2 and ITGA1 on cancer cells highly expressing Grb2 through suppressing ERK phosphorylation

    doi:

    Figure Lengend Snippet: Synergistic effect of Grb2 and ITGA1 inhibition through ERK signaling. A. Hela cells and Jurkat cells were treated with Grb2 siRNA in the absence or presence of ITGA1 siRNA. Western blot analysis for expression of phosphorylated and total ERK, cleaved caspase-3 and cleaved PARP. B. The percentages of the sub-G0/G1 population were determined by flow cytometry in Hela cells and Jurkat cells. Data are represented as mean ± S.E.M. (n=3). *** P

    Article Snippet: The membranes were incubated overnight with specific Grb2 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), ITGA1 (Abcam), ERK (Cell Signaling Technology), phosphor-ERK (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology) and cleaved PARP (Cell Signaling Technology) were exposed to secondary antibodies coupled to horseradish peroxidase for 2 hours at room temperature.

    Techniques: Inhibition, Western Blot, Expressing, Flow Cytometry, Cytometry

    ITGA1 and ERK expression patterns after treatment with Grb2 siRNA or negative control siRNA in Hela cells and Jurkat cells. A. ITGA1 and ERK expression in Hela cells treating with Grb2 siRNA or NC siRNA. B. ITGA1 and ERK expression in Jurkat cells treating with Grb2 siRNA or NC siRNA. Western blot analysis was performed to detect the expression level of ITGA1, phosphorylated and total ERK protein.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Synergistic anticancer effect of Grb2 and ITGA1 on cancer cells highly expressing Grb2 through suppressing ERK phosphorylation

    doi:

    Figure Lengend Snippet: ITGA1 and ERK expression patterns after treatment with Grb2 siRNA or negative control siRNA in Hela cells and Jurkat cells. A. ITGA1 and ERK expression in Hela cells treating with Grb2 siRNA or NC siRNA. B. ITGA1 and ERK expression in Jurkat cells treating with Grb2 siRNA or NC siRNA. Western blot analysis was performed to detect the expression level of ITGA1, phosphorylated and total ERK protein.

    Article Snippet: The membranes were incubated overnight with specific Grb2 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), ITGA1 (Abcam), ERK (Cell Signaling Technology), phosphor-ERK (Cell Signaling Technology), cleaved caspase-3 (Cell Signaling Technology) and cleaved PARP (Cell Signaling Technology) were exposed to secondary antibodies coupled to horseradish peroxidase for 2 hours at room temperature.

    Techniques: Expressing, Negative Control, Western Blot

    Induction of phosphorylated ERK in ROS-exposed lymphocytes. PBMCs were treated with H 2 O 2 (500 µM) (filled circle) or PBS (open square) at 37°C and analyzed for pERK by flow cytometry at indicated time points. Graphs show the percentage of pERK-positive cells ( A ) and pERK MFI ( B ) in gated lymphocytes (mean ± SEM, results from 4–6 donors). ( C ) Lymphocytes were treated with 250 µM H 2 O 2 (1–10 min), PBS (10 min) or 50 ng/ml PMA (40 min) at 37°C. pERK1/2 was detected in whole cell lysates by Western blot. Total ERK1/2 was measured as loading control in parallel wells. A representative blot of 3 is shown. D. Inhibition of pERK formation by the ERK1/2 pathway inhibitor (PD98059, 25 µM) in gated lymphocytes after exposure to 500 µM H 2 O 2 (10 min) shown as percent positive cells. E. Inhibition of pERK by PD98059 in NK cells exposed to PMA-stimulated monocytes shown as percent positive cells. Panels D and E are the mean ± SEM of results obtained using 5 donors. *P

    Journal: PLoS ONE

    Article Title: Role of the ERK Pathway for Oxidant-Induced Parthanatos in Human Lymphocytes

    doi: 10.1371/journal.pone.0089646

    Figure Lengend Snippet: Induction of phosphorylated ERK in ROS-exposed lymphocytes. PBMCs were treated with H 2 O 2 (500 µM) (filled circle) or PBS (open square) at 37°C and analyzed for pERK by flow cytometry at indicated time points. Graphs show the percentage of pERK-positive cells ( A ) and pERK MFI ( B ) in gated lymphocytes (mean ± SEM, results from 4–6 donors). ( C ) Lymphocytes were treated with 250 µM H 2 O 2 (1–10 min), PBS (10 min) or 50 ng/ml PMA (40 min) at 37°C. pERK1/2 was detected in whole cell lysates by Western blot. Total ERK1/2 was measured as loading control in parallel wells. A representative blot of 3 is shown. D. Inhibition of pERK formation by the ERK1/2 pathway inhibitor (PD98059, 25 µM) in gated lymphocytes after exposure to 500 µM H 2 O 2 (10 min) shown as percent positive cells. E. Inhibition of pERK by PD98059 in NK cells exposed to PMA-stimulated monocytes shown as percent positive cells. Panels D and E are the mean ± SEM of results obtained using 5 donors. *P

    Article Snippet: For detection of phosphorylated ERK (p-ERK), membranes were incubated with rabbit anti phospho-ERK1/2 Ab (Cell Signaling Technology, MA, USA) or rabbit total ERK1/2 (Cell signaling technology) followed by incubation with a HRP-conjugated goat anti-rabbit Ab (DAKO).

    Techniques: Flow Cytometry, Cytometry, Western Blot, Inhibition

    Inhibition of ERK phosphorylation in lymphocytes exposed to oxygen radicals by ERK pathway inhibitor but not by PARP inhibitor. PBMCs or NK cells were preincubated in presence or absence of ERK1/2 inhibitor PD98059 (25 µM) or PARP-1 inhibitor PJ34 (2 µM) for 1 h at 37°C. ( A ) A Representative dot plots of pERK + NK cells after 10 min exposure to PMA-stimulated monocytes . is shown. ( B ) Mean ± SEM of pERK positive cells in gated lymphocytes after 10 min exposure to H 2 O 2 . ( C ) Mean ± SEM of pERK positive NK cells after 10 min exposure to PMA-stimulated monocytes. B–C : mean ± SEM of 4–6 experiments. *P

    Journal: PLoS ONE

    Article Title: Role of the ERK Pathway for Oxidant-Induced Parthanatos in Human Lymphocytes

    doi: 10.1371/journal.pone.0089646

    Figure Lengend Snippet: Inhibition of ERK phosphorylation in lymphocytes exposed to oxygen radicals by ERK pathway inhibitor but not by PARP inhibitor. PBMCs or NK cells were preincubated in presence or absence of ERK1/2 inhibitor PD98059 (25 µM) or PARP-1 inhibitor PJ34 (2 µM) for 1 h at 37°C. ( A ) A Representative dot plots of pERK + NK cells after 10 min exposure to PMA-stimulated monocytes . is shown. ( B ) Mean ± SEM of pERK positive cells in gated lymphocytes after 10 min exposure to H 2 O 2 . ( C ) Mean ± SEM of pERK positive NK cells after 10 min exposure to PMA-stimulated monocytes. B–C : mean ± SEM of 4–6 experiments. *P

    Article Snippet: For detection of phosphorylated ERK (p-ERK), membranes were incubated with rabbit anti phospho-ERK1/2 Ab (Cell Signaling Technology, MA, USA) or rabbit total ERK1/2 (Cell signaling technology) followed by incubation with a HRP-conjugated goat anti-rabbit Ab (DAKO).

    Techniques: Inhibition

    Oxidant-induced poly ADP-ribose accumulation: role of the ERK pathway. ( A–B ) Lymphocytes were preincubated with or without the ERK1/2 inhibitor PD98059 (25 µM) or the PARP-1 inhibitor PJ34 (2 µM) for 1 hr at 37°C before exposure to 500 µM H 2 O 2 for 20 min. PAR accumulation was analyzed in whole cell lysates. ( A ) Representative Western blot and ( B ) mean ± SEM of results from 4 donors (O.D., optical density). β-tubulin was utilized as loading control and H 2 O 2 -treated HELA cells served as positive controls. ( C ) Flow cytometry analysis of PAR accumulation following exposure to H 2 O 2 (500 µM, filled circle) or PBS (open square). ( D ) Inhibition of PAR formation in lymphocytes after preincubation with PD98059 (25 µM) or PJ34 (2 µM). PAR accumulation was measured after 20 min exposure to 500 µM H 2 O 2 by flow cytometry. Data are the MFI of PAR in gated lymphocytes (mean ± SEM of results obtained in 4–6 donors) *P

    Journal: PLoS ONE

    Article Title: Role of the ERK Pathway for Oxidant-Induced Parthanatos in Human Lymphocytes

    doi: 10.1371/journal.pone.0089646

    Figure Lengend Snippet: Oxidant-induced poly ADP-ribose accumulation: role of the ERK pathway. ( A–B ) Lymphocytes were preincubated with or without the ERK1/2 inhibitor PD98059 (25 µM) or the PARP-1 inhibitor PJ34 (2 µM) for 1 hr at 37°C before exposure to 500 µM H 2 O 2 for 20 min. PAR accumulation was analyzed in whole cell lysates. ( A ) Representative Western blot and ( B ) mean ± SEM of results from 4 donors (O.D., optical density). β-tubulin was utilized as loading control and H 2 O 2 -treated HELA cells served as positive controls. ( C ) Flow cytometry analysis of PAR accumulation following exposure to H 2 O 2 (500 µM, filled circle) or PBS (open square). ( D ) Inhibition of PAR formation in lymphocytes after preincubation with PD98059 (25 µM) or PJ34 (2 µM). PAR accumulation was measured after 20 min exposure to 500 µM H 2 O 2 by flow cytometry. Data are the MFI of PAR in gated lymphocytes (mean ± SEM of results obtained in 4–6 donors) *P

    Article Snippet: For detection of phosphorylated ERK (p-ERK), membranes were incubated with rabbit anti phospho-ERK1/2 Ab (Cell Signaling Technology, MA, USA) or rabbit total ERK1/2 (Cell signaling technology) followed by incubation with a HRP-conjugated goat anti-rabbit Ab (DAKO).

    Techniques: Western Blot, Flow Cytometry, Cytometry, Inhibition

    Protection of lymphocytes from ROS-induced apoptosis by an ERK pathway inhibitor. MACS-purified human CD8 + T cells or NK cells were preincubated with the ERK1/2 inhibitor PD98059 (25 µM) (filled triangle) for 1 h at 37°C. The T cells and NK cells were then incubated overnight in the presence of PD98059 with H 2 O 2 at indicated concentrations ( A–B ) or with ROS-producing monocytes (MØ) at indicated MØ:NK ratios ( C–D ). Lymphocyte viability was assessed using the Live/Dead Fixable Violet Dead Cell Stain kit. ERK inhibitor-equivalent concentrations of DMSO were used as control (open square). Results obtained using DMSO did not significantly differ from PBS. Data are the mean ± SEM of results obtained using blood from 3–7 donors. *P

    Journal: PLoS ONE

    Article Title: Role of the ERK Pathway for Oxidant-Induced Parthanatos in Human Lymphocytes

    doi: 10.1371/journal.pone.0089646

    Figure Lengend Snippet: Protection of lymphocytes from ROS-induced apoptosis by an ERK pathway inhibitor. MACS-purified human CD8 + T cells or NK cells were preincubated with the ERK1/2 inhibitor PD98059 (25 µM) (filled triangle) for 1 h at 37°C. The T cells and NK cells were then incubated overnight in the presence of PD98059 with H 2 O 2 at indicated concentrations ( A–B ) or with ROS-producing monocytes (MØ) at indicated MØ:NK ratios ( C–D ). Lymphocyte viability was assessed using the Live/Dead Fixable Violet Dead Cell Stain kit. ERK inhibitor-equivalent concentrations of DMSO were used as control (open square). Results obtained using DMSO did not significantly differ from PBS. Data are the mean ± SEM of results obtained using blood from 3–7 donors. *P

    Article Snippet: For detection of phosphorylated ERK (p-ERK), membranes were incubated with rabbit anti phospho-ERK1/2 Ab (Cell Signaling Technology, MA, USA) or rabbit total ERK1/2 (Cell signaling technology) followed by incubation with a HRP-conjugated goat anti-rabbit Ab (DAKO).

    Techniques: Magnetic Cell Separation, Purification, Incubation, Staining

    Morphine activated ERKs via G protein-dependent pathway (A and B), 1 µM PKC inhibitor Ro-31-8425, or DMSO (control) was used to pretreat the cells for 1.5 h, and then 1 µM morphine (A) or etorphine (B) was added for various times as indicated. The samples were subjected to immunoblotting for ERK phosphorylation. C, HEK293 cells were treated with 1 µM PKC inhibitor Ro-31-8425 or DMSO (control) for 1.5 h followed by a 10-min treatment of 1 µM agonists. PKC activities were determined with immunoblotting using antibody specific for PKC phosphorylated substrates. *, p

    Journal: Molecular pharmacology

    Article Title: ?-Arrestin-Dependent ?-Opioid Receptor-Activated Extracellular Signal-Regulated Kinases (ERKs) Translocate to Nucleus in Contrast to G Protein-Dependent ERK Activation

    doi: 10.1124/mol.107.039842

    Figure Lengend Snippet: Morphine activated ERKs via G protein-dependent pathway (A and B), 1 µM PKC inhibitor Ro-31-8425, or DMSO (control) was used to pretreat the cells for 1.5 h, and then 1 µM morphine (A) or etorphine (B) was added for various times as indicated. The samples were subjected to immunoblotting for ERK phosphorylation. C, HEK293 cells were treated with 1 µM PKC inhibitor Ro-31-8425 or DMSO (control) for 1.5 h followed by a 10-min treatment of 1 µM agonists. PKC activities were determined with immunoblotting using antibody specific for PKC phosphorylated substrates. *, p

    Article Snippet: Because β-arrestin has been shown to translocate into the nucleus ( ; ; ), it is reasonable to propose that the β-arrestin-ERK complex is the cause for the observed increase in the phosphorylated ERKs within the nucleus fractions after etorphine treatment.

    Techniques:

    . Immunoblotting was used to monitor the phosphorylated ERK level, and antibodies to β-actin, Rab4, and Histone3 were used to examine the success of separating nucleus from cytoplasm. m10 and m20 represents phosphorylated ERKs level 10 and 20 min after morphine treatment, respectively; e10 and e20 represents phosphorylated ERK levels 10 and 20 min after etorphine treatment, respectively. B, different concentrations of agonists were added for 10 min. The level of phosphorylated ERKs in the nucleus was measured as in A. C, HEK293 cells were transfected with 1 µg of β-arrestin2 or Dynamin K44E or vector (control). Then 1 µM agonists were added for 10 min. After nucleus extraction, the level of phosphorylated ERKs in the nucleus was determined by immunoblotting. *, p

    Journal: Molecular pharmacology

    Article Title: ?-Arrestin-Dependent ?-Opioid Receptor-Activated Extracellular Signal-Regulated Kinases (ERKs) Translocate to Nucleus in Contrast to G Protein-Dependent ERK Activation

    doi: 10.1124/mol.107.039842

    Figure Lengend Snippet: . Immunoblotting was used to monitor the phosphorylated ERK level, and antibodies to β-actin, Rab4, and Histone3 were used to examine the success of separating nucleus from cytoplasm. m10 and m20 represents phosphorylated ERKs level 10 and 20 min after morphine treatment, respectively; e10 and e20 represents phosphorylated ERK levels 10 and 20 min after etorphine treatment, respectively. B, different concentrations of agonists were added for 10 min. The level of phosphorylated ERKs in the nucleus was measured as in A. C, HEK293 cells were transfected with 1 µg of β-arrestin2 or Dynamin K44E or vector (control). Then 1 µM agonists were added for 10 min. After nucleus extraction, the level of phosphorylated ERKs in the nucleus was determined by immunoblotting. *, p

    Article Snippet: Because β-arrestin has been shown to translocate into the nucleus ( ; ; ), it is reasonable to propose that the β-arrestin-ERK complex is the cause for the observed increase in the phosphorylated ERKs within the nucleus fractions after etorphine treatment.

    Techniques: Transfection, Plasmid Preparation

    ATLa prevents ATP-evoked ERK and JNK phosphorylation in primary astrocyte cultures. (A) Representative images demonstrating that ALXR colocalizes with the astrocyte marker GFAP in cultured primary astrocytes. Bar, 50 μm. (B) Western blots probed for phosphorylated ERK and JNK in samples from primary astrocytes stimulated with ATP, SP, IL-1β, and TNF-α for 15 min. Incubation with 10 nM ATLa, starting 30 min before TNF-α stimulation, had no effect on JNK phosphorylation (C), whereas ATLa prevented both ERK and JNK phosphorylation evoked by ATP (D and E). Each bar represents the mean ± SEM ( n = 4–5). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Lipoxins and aspirin-triggered lipoxin inhibit inflammatory pain processing

    doi: 10.1084/jem.20061826

    Figure Lengend Snippet: ATLa prevents ATP-evoked ERK and JNK phosphorylation in primary astrocyte cultures. (A) Representative images demonstrating that ALXR colocalizes with the astrocyte marker GFAP in cultured primary astrocytes. Bar, 50 μm. (B) Western blots probed for phosphorylated ERK and JNK in samples from primary astrocytes stimulated with ATP, SP, IL-1β, and TNF-α for 15 min. Incubation with 10 nM ATLa, starting 30 min before TNF-α stimulation, had no effect on JNK phosphorylation (C), whereas ATLa prevented both ERK and JNK phosphorylation evoked by ATP (D and E). Each bar represents the mean ± SEM ( n = 4–5). *, P

    Article Snippet: Membranes were incubated with antibodies against ALXR (1:2,000; Biologicals), phosphorylated JNK (P-JNK), total JNK, phosphorylated (P-ERK), and total ERK (1:10,000; Cell Signaling).

    Techniques: Marker, Cell Culture, Western Blot, Incubation

    ITF-2 induction by activation of Wnt signaling pathway ( A ) Western blot analysis for Wnt/β-catenin signaling proteins demonstrated that dephosphorylated β-catenin was translocated to the nucleus and phosphorylated Ser9 of GSK3β was accumulated in cytosol fraction in AZD6244 resistant cell lines (M14/AZD-3 and LOX-IMVI). ( B ) Western blot analysis showed that the p90RSK was downregulated in AZD6244 resistant cell lines. ( C ) GST pull-down assay using pGEX-GSK3β and Glutathione-Sepharose 4B (GST) beads demonstrated the direct interaction of p-ERK and GSK3β and phosphorylation of GSK3β at Ser9 in AZD6244 resistant cell lines (M14/AZD-3 and LOX-IMVI).

    Journal: Oncotarget

    Article Title: Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells

    doi: 10.18632/oncotarget.17866

    Figure Lengend Snippet: ITF-2 induction by activation of Wnt signaling pathway ( A ) Western blot analysis for Wnt/β-catenin signaling proteins demonstrated that dephosphorylated β-catenin was translocated to the nucleus and phosphorylated Ser9 of GSK3β was accumulated in cytosol fraction in AZD6244 resistant cell lines (M14/AZD-3 and LOX-IMVI). ( B ) Western blot analysis showed that the p90RSK was downregulated in AZD6244 resistant cell lines. ( C ) GST pull-down assay using pGEX-GSK3β and Glutathione-Sepharose 4B (GST) beads demonstrated the direct interaction of p-ERK and GSK3β and phosphorylation of GSK3β at Ser9 in AZD6244 resistant cell lines (M14/AZD-3 and LOX-IMVI).

    Article Snippet: Specific antibodies were as follows: ITF-2 (Abcam, Cambridge, MA), phospho-beta-catenin (p-beta-catenin), beta-catenin, phospho-ERK (p-ERK), ERK, phospho-GSK3 beta (Y216 and S9), GSK-3 alpha and beta, phospho-90RSK (Y573), 90RSK, PARP, cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), dephospho-beta-catenin (de-p-beta-catenin) (Enzo bioscience Inc. France), KRAS, B-RAF, phosphor-MEK, MEK, and beta-actin (Sigma-Aldrich, St. Louis, MO).

    Techniques: Activation Assay, Western Blot, Pull Down Assay

    Establishment of an acquired AZD6244 resistant cell line, M14/AZD-3 and overexpression of ITF-2 in M14/AZD-3 ( A ) M14/AZD-3 cell line was established by long-term treatment of an AZD6244 sensitive melanoma cell line, M14, with increasing dose of AZD6244. ( B ) With 1 μM of AZD6244, suppression of phosphorylated ERK (p-ERK) and cleavage of poly (ADP-ribose) polymerase (PARP) did occur in M14, but not in M13/AZD-3 (acquired AZD6244 resistant cell line) and LOX-IMVI (primary AZD6244 resistant cell line). ( C ) Overexpression of ITF-2 protein in M14/AZD-3 and LOX-IMVI.

    Journal: Oncotarget

    Article Title: Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells

    doi: 10.18632/oncotarget.17866

    Figure Lengend Snippet: Establishment of an acquired AZD6244 resistant cell line, M14/AZD-3 and overexpression of ITF-2 in M14/AZD-3 ( A ) M14/AZD-3 cell line was established by long-term treatment of an AZD6244 sensitive melanoma cell line, M14, with increasing dose of AZD6244. ( B ) With 1 μM of AZD6244, suppression of phosphorylated ERK (p-ERK) and cleavage of poly (ADP-ribose) polymerase (PARP) did occur in M14, but not in M13/AZD-3 (acquired AZD6244 resistant cell line) and LOX-IMVI (primary AZD6244 resistant cell line). ( C ) Overexpression of ITF-2 protein in M14/AZD-3 and LOX-IMVI.

    Article Snippet: Specific antibodies were as follows: ITF-2 (Abcam, Cambridge, MA), phospho-beta-catenin (p-beta-catenin), beta-catenin, phospho-ERK (p-ERK), ERK, phospho-GSK3 beta (Y216 and S9), GSK-3 alpha and beta, phospho-90RSK (Y573), 90RSK, PARP, cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), dephospho-beta-catenin (de-p-beta-catenin) (Enzo bioscience Inc. France), KRAS, B-RAF, phosphor-MEK, MEK, and beta-actin (Sigma-Aldrich, St. Louis, MO).

    Techniques: Over Expression

    Model of the ITF-2 induction via ERK/GSK3β/β-catenin In AZD6244 resistant cells, p-ERK directly interacts with GSK3β, leading to phosphorylation at Ser9 and dephosphorylation of β-catenin. The dephosphorylated β-catenin is translocated to the nucleus and the association of β-catenin and T-cell factor results in the transcription of ITF-2 gene, one of Wnt target genes.

    Journal: Oncotarget

    Article Title: Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells

    doi: 10.18632/oncotarget.17866

    Figure Lengend Snippet: Model of the ITF-2 induction via ERK/GSK3β/β-catenin In AZD6244 resistant cells, p-ERK directly interacts with GSK3β, leading to phosphorylation at Ser9 and dephosphorylation of β-catenin. The dephosphorylated β-catenin is translocated to the nucleus and the association of β-catenin and T-cell factor results in the transcription of ITF-2 gene, one of Wnt target genes.

    Article Snippet: Specific antibodies were as follows: ITF-2 (Abcam, Cambridge, MA), phospho-beta-catenin (p-beta-catenin), beta-catenin, phospho-ERK (p-ERK), ERK, phospho-GSK3 beta (Y216 and S9), GSK-3 alpha and beta, phospho-90RSK (Y573), 90RSK, PARP, cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), dephospho-beta-catenin (de-p-beta-catenin) (Enzo bioscience Inc. France), KRAS, B-RAF, phosphor-MEK, MEK, and beta-actin (Sigma-Aldrich, St. Louis, MO).

    Techniques: De-Phosphorylation Assay

    Basal levels of ITF-2 in AZD6244 resistant and sensitive cell lines ( A ) Relative expression of endogenous ITF-2 mRNA in each indicated cell line based on the expression in SW620. Each sample was tested in triplicate, and gene expression levels were normalized to those of β-actin mRNA. The mRNA levels of ITF-2 in AZD6244 resistant cell lines were significantly higher than those in sensitive cell lines. Statistical significances are calculated by the Mann-Whitney U -test. ( B ) Equal amounts of total cellular proteins were subjected to western blot analysis for ITF-2, and phospho-specific and total ERK1/2. ITF-2 was elevated in AZD6244 resistant cell lines except SNB-19, but the p-ERK levels were not significantly different according to the sensitivity to AZD6244. Beta-actin was included as a loading control.

    Journal: Oncotarget

    Article Title: Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells

    doi: 10.18632/oncotarget.17866

    Figure Lengend Snippet: Basal levels of ITF-2 in AZD6244 resistant and sensitive cell lines ( A ) Relative expression of endogenous ITF-2 mRNA in each indicated cell line based on the expression in SW620. Each sample was tested in triplicate, and gene expression levels were normalized to those of β-actin mRNA. The mRNA levels of ITF-2 in AZD6244 resistant cell lines were significantly higher than those in sensitive cell lines. Statistical significances are calculated by the Mann-Whitney U -test. ( B ) Equal amounts of total cellular proteins were subjected to western blot analysis for ITF-2, and phospho-specific and total ERK1/2. ITF-2 was elevated in AZD6244 resistant cell lines except SNB-19, but the p-ERK levels were not significantly different according to the sensitivity to AZD6244. Beta-actin was included as a loading control.

    Article Snippet: Specific antibodies were as follows: ITF-2 (Abcam, Cambridge, MA), phospho-beta-catenin (p-beta-catenin), beta-catenin, phospho-ERK (p-ERK), ERK, phospho-GSK3 beta (Y216 and S9), GSK-3 alpha and beta, phospho-90RSK (Y573), 90RSK, PARP, cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), dephospho-beta-catenin (de-p-beta-catenin) (Enzo bioscience Inc. France), KRAS, B-RAF, phosphor-MEK, MEK, and beta-actin (Sigma-Aldrich, St. Louis, MO).

    Techniques: Expressing, MANN-WHITNEY, Western Blot

    ERK activation by CXCR4 occurs independently of the ability of the CXCR4 to be ubiquitinated. A, ERK activation was evaluated in HEK293 cells transiently transfected with HA-WT CXCR4 or HA-3K/R CXCR4 and stimulated with CXCL12 (10 n m ) for 0, 5, 15, 30, or 60 min, and ERK activity in cell lysates assessed was as described under “Experimental Procedures.” B , quantitation of ERK activation was based on the amount of ERK detected using an anti-P-ERK antibody; total ERK, assessed using the ERK2 antibody, was indistinguishable in all conditions, and thus the data were not normalized to total ERK. Data are mean ± S.E. from three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation *

    doi: 10.1074/jbc.M808507200

    Figure Lengend Snippet: ERK activation by CXCR4 occurs independently of the ability of the CXCR4 to be ubiquitinated. A, ERK activation was evaluated in HEK293 cells transiently transfected with HA-WT CXCR4 or HA-3K/R CXCR4 and stimulated with CXCL12 (10 n m ) for 0, 5, 15, 30, or 60 min, and ERK activity in cell lysates assessed was as described under “Experimental Procedures.” B , quantitation of ERK activation was based on the amount of ERK detected using an anti-P-ERK antibody; total ERK, assessed using the ERK2 antibody, was indistinguishable in all conditions, and thus the data were not normalized to total ERK. Data are mean ± S.E. from three independent experiments.

    Article Snippet: Thus, we assessed ERK phosphorylation in response to CXCL12 activation of WT and 3K/R CXCR4 using a p-ERK p42/p44 Thr/Tyr antibody (Cell Signaling Technologies).

    Techniques: Activation Assay, Transfection, Activity Assay, Quantitation Assay

    The selective activation of MAPK phosphorylation induced by different magnitudes of tensile strain. Western blot of MAPK was shown as p-ERK and ERK ( a ), p-p38 and p38 ( b ), and p-JNK and JNK ( c ). d Quantification graph for the protein levels of the phosphorylated

    Journal: International Orthopaedics

    Article Title: Periprosthetic strain magnitude-dependent upregulation of type I collagen synthesis in human osteoblasts through an ERK1/2 pathway

    doi: 10.1007/s00264-009-0735-z

    Figure Lengend Snippet: The selective activation of MAPK phosphorylation induced by different magnitudes of tensile strain. Western blot of MAPK was shown as p-ERK and ERK ( a ), p-p38 and p38 ( b ), and p-JNK and JNK ( c ). d Quantification graph for the protein levels of the phosphorylated

    Article Snippet: After being blocked with 5% skim milk for two hours at room temperature, the membranes were probed overnight at 4°C with anti-ERK, anti-JNK, anti-p38, anti-phospho-p38, anti-phospho-ERK, and anti-phospho-JNK (Cell Signaling, USA).

    Techniques: Activation Assay, Western Blot

    Heparan sulfate induces an apoptosis/necroptosis signal pathway.  (A)  HS fragments are cleaved by heparanase from a HS proteoglycan, which is localized on the plasma membrane of endothelial cells.  (B)  Structure of HS proteoglycan.  (C)  HS interacts with a pattern recognition receptor (i.e., toll-like receptor 4) localized on the cell surface of cardiomyocytes and activates a pro-apoptotic intrinsic pathway. The signaling cascade involves phosphorylation of ERK 1/2 resulting in the release of mitochondrial cytochrome C, which leads to cleavage and activation of caspase 3. In the next step of this pathway, PARP is cleaved and deactivated by activated caspase 3. Induction of TNF-α caused by HS inhibits the pro-apoptotic pathway and induces phosphorylation of RIP3 and necroptosis. These apoptosis and necroptosis signal pathways represent our Petri net model. White circles are places, and black rectangles are transitions. Unidirectional arcs indicate directed flows. Bidirectional arcs indicate read arcs, which influence transitions, but do not consume tokens. Modified from Martin et al., Sarrazin et al., and Maeda (  13 –  15 ). HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; ERK, extracellular signal-regulated kinase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha; Ser, serine; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylgalactosamine; IdoA, iduronic acid.

    Journal: Frontiers in Immunology

    Article Title: Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning

    doi: 10.3389/fimmu.2018.00393

    Figure Lengend Snippet: Heparan sulfate induces an apoptosis/necroptosis signal pathway. (A) HS fragments are cleaved by heparanase from a HS proteoglycan, which is localized on the plasma membrane of endothelial cells. (B) Structure of HS proteoglycan. (C) HS interacts with a pattern recognition receptor (i.e., toll-like receptor 4) localized on the cell surface of cardiomyocytes and activates a pro-apoptotic intrinsic pathway. The signaling cascade involves phosphorylation of ERK 1/2 resulting in the release of mitochondrial cytochrome C, which leads to cleavage and activation of caspase 3. In the next step of this pathway, PARP is cleaved and deactivated by activated caspase 3. Induction of TNF-α caused by HS inhibits the pro-apoptotic pathway and induces phosphorylation of RIP3 and necroptosis. These apoptosis and necroptosis signal pathways represent our Petri net model. White circles are places, and black rectangles are transitions. Unidirectional arcs indicate directed flows. Bidirectional arcs indicate read arcs, which influence transitions, but do not consume tokens. Modified from Martin et al., Sarrazin et al., and Maeda ( 13 – 15 ). HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; ERK, extracellular signal-regulated kinase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha; Ser, serine; Xyl, xylose; Gal, galactose; GlcNAc, N-acetylgalactosamine; IdoA, iduronic acid.

    Article Snippet: After blocking, membranes were incubated with specific primary antibodies against caspase 3 (Cell Signaling, Danvers, MA, USA), poly-(ADP-ribose) polymerase (PARP) (Cell Signaling), extracellular signal-regulated kinase (ERK) 1/2 (Cell Signaling), phospho-ERK 1/2 (Cell Signaling), cytochrome C (Cell Signaling), RIP3 (Bio-Rad), phospho-RIP3 (Ser232) (Abcam), MLKL (Cell Signaling), phospho-MLKL (Ser345) (Cell Signaling), and vinculin (Sigma).

    Techniques: Activation Assay, Modification

    Simulated time course of all involved components of the apoptosis/necroptosis signal pathway induced by HS. Simulated time course of the components involved in intrinsic apoptosis and necroptosis signaling pathways for cardiomyocytes exposed to three different HS concentrations [5 (green), 10 (blue), and 20 (purple) μg/ml of HS]. The blue background represents the standard error of the measured data. The model was simulated based on relative cleaved PARP values. The filled points in graphs represent data used for training and the unfilled for validation. Simulated time course for relative protein expression of  (A)  phosphorylation of ERK 1/2,  (B)  cytochrome C,  (C)  cleaved caspase 3,  (D)  cleaved PARP, and  (E)  phosphorylation of RIP3 is shown.  (F)  Simulated data of relative TNF-α mRNA expression. HS, heparan sulfate; ph-ERK, phospho-extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha.

    Journal: Frontiers in Immunology

    Article Title: Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning

    doi: 10.3389/fimmu.2018.00393

    Figure Lengend Snippet: Simulated time course of all involved components of the apoptosis/necroptosis signal pathway induced by HS. Simulated time course of the components involved in intrinsic apoptosis and necroptosis signaling pathways for cardiomyocytes exposed to three different HS concentrations [5 (green), 10 (blue), and 20 (purple) μg/ml of HS]. The blue background represents the standard error of the measured data. The model was simulated based on relative cleaved PARP values. The filled points in graphs represent data used for training and the unfilled for validation. Simulated time course for relative protein expression of (A) phosphorylation of ERK 1/2, (B) cytochrome C, (C) cleaved caspase 3, (D) cleaved PARP, and (E) phosphorylation of RIP3 is shown. (F) Simulated data of relative TNF-α mRNA expression. HS, heparan sulfate; ph-ERK, phospho-extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase; RIP, receptor-interacting protein; TNF-α, tumor necrosis factor alpha.

    Article Snippet: After blocking, membranes were incubated with specific primary antibodies against caspase 3 (Cell Signaling, Danvers, MA, USA), poly-(ADP-ribose) polymerase (PARP) (Cell Signaling), extracellular signal-regulated kinase (ERK) 1/2 (Cell Signaling), phospho-ERK 1/2 (Cell Signaling), cytochrome C (Cell Signaling), RIP3 (Bio-Rad), phospho-RIP3 (Ser232) (Abcam), MLKL (Cell Signaling), phospho-MLKL (Ser345) (Cell Signaling), and vinculin (Sigma).

    Techniques: Expressing

    Heparan sulfate induces a pro-apoptotic pathway. HL-1 cells exposed to 10 μg/ml HS for 16 h showed a significant increase in protein expression of  (A)  phospho-ERK 1/2,  (B)  cytochrome C,  (C) cleaved  PARP, and  (D) cleaved  caspase 3, compared to unstimulated cells. Protein expression was normalized to unstimulated cells.  (E)  Relative mRNA expressions of HL-1 cells exposed to HS were analyzed by quantitative real-time PCR, compared to unstimulated cells. Caspase 3 mRNA expression was normalized to reference gene S7 and unstimulated cells.  (F)  Relative caspase 3 activity of cardiomyocytes exposed to HS, compared to unstimulated cells. The data represent the mean ± SD of triplicate samples for three independent experiments. HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; p-ERK, phospho-extracellular signal-regulated kinase; statistical significance was performed by using unpaired  t -test. * p

    Journal: Frontiers in Immunology

    Article Title: Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning

    doi: 10.3389/fimmu.2018.00393

    Figure Lengend Snippet: Heparan sulfate induces a pro-apoptotic pathway. HL-1 cells exposed to 10 μg/ml HS for 16 h showed a significant increase in protein expression of (A) phospho-ERK 1/2, (B) cytochrome C, (C) cleaved PARP, and (D) cleaved caspase 3, compared to unstimulated cells. Protein expression was normalized to unstimulated cells. (E) Relative mRNA expressions of HL-1 cells exposed to HS were analyzed by quantitative real-time PCR, compared to unstimulated cells. Caspase 3 mRNA expression was normalized to reference gene S7 and unstimulated cells. (F) Relative caspase 3 activity of cardiomyocytes exposed to HS, compared to unstimulated cells. The data represent the mean ± SD of triplicate samples for three independent experiments. HS, heparan sulfate; PARP, poly-(ADP-ribose) polymerase; p-ERK, phospho-extracellular signal-regulated kinase; statistical significance was performed by using unpaired t -test. * p

    Article Snippet: After blocking, membranes were incubated with specific primary antibodies against caspase 3 (Cell Signaling, Danvers, MA, USA), poly-(ADP-ribose) polymerase (PARP) (Cell Signaling), extracellular signal-regulated kinase (ERK) 1/2 (Cell Signaling), phospho-ERK 1/2 (Cell Signaling), cytochrome C (Cell Signaling), RIP3 (Bio-Rad), phospho-RIP3 (Ser232) (Abcam), MLKL (Cell Signaling), phospho-MLKL (Ser345) (Cell Signaling), and vinculin (Sigma).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay

    Effect of for 1 on EGF-induced activation of EGFR in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), p-ERK (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P

    Journal: BioMed Research International

    Article Title: Degalactotigonin, a Steroidal Glycoside from Solanum nigrum, Induces Apoptosis and Cell Cycle Arrest via Inhibiting the EGFR Signaling Pathways in Pancreatic Cancer Cells

    doi: 10.1155/2018/3120972

    Figure Lengend Snippet: Effect of for 1 on EGF-induced activation of EGFR in PANC1 cells. (a, b, and c) Serum-starved PANC1 cells were stimulated with EGF (100 ng/mL) in the presence of the indicted concentrations of 1 for 5 min. Whole cell lysates were prepared and Western blotting was performed to determine the expression level of p-EGFR (a), EGFR (a), p-Akt (b), Akt (b), p-ERK (c), and ERK (c). α -Tubulin was used as a loading control. The blots were quantified by Image J software and the levels of p-EGFR, p-Akt, and p-ERK (normalized to EGFR, Akt, and ERK, respectively) were expressed as the mean ± SD of three independent experiments. ∗ P

    Article Snippet: Anti-phospho-EGFR, anti-Akt, anti-phospho Akt (Ser473), anti-ERK, and anti-phospho-ERK antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Western Blot, Expressing, Software

    ADAM-17 activity is ERK dependent. PD98059 (PD) inhibits 5-HT-induced ADAM-17 activation measured by cleavage of the quenched fluorogenic peptide substrate MCA-PLAQAV(Dpa). Figure shows data from 4 experiments performed in triplicates.

    Journal: The American journal of the medical sciences

    Article Title: ADAM-17 is activated by the Mitogenic Protein Kinase ERK in a Model of Kidney Fibrosis

    doi: 10.1097/MAJ.0b013e3181cb4487

    Figure Lengend Snippet: ADAM-17 activity is ERK dependent. PD98059 (PD) inhibits 5-HT-induced ADAM-17 activation measured by cleavage of the quenched fluorogenic peptide substrate MCA-PLAQAV(Dpa). Figure shows data from 4 experiments performed in triplicates.

    Article Snippet: Cells were cellular proteins were immunoprecipitated using ADAM-17 antibody (QED) or phospho-ERK antibody (Cell Signaling) employing standard immunoprecipiation protocol .

    Techniques: Activity Assay, Activation Assay

    ADAM-17 phosphorylation is ERK dependent. A) PD98059 (PD) inhibits 5-HT-induced ADAM-17 threonine phosphorylation (p-Threonine). B) Phosphorylated ERK interacts with ADAM-17 during 5-HT treatment in mesangial cells. IP: immunoprecipitation. Figure shows

    Journal: The American journal of the medical sciences

    Article Title: ADAM-17 is activated by the Mitogenic Protein Kinase ERK in a Model of Kidney Fibrosis

    doi: 10.1097/MAJ.0b013e3181cb4487

    Figure Lengend Snippet: ADAM-17 phosphorylation is ERK dependent. A) PD98059 (PD) inhibits 5-HT-induced ADAM-17 threonine phosphorylation (p-Threonine). B) Phosphorylated ERK interacts with ADAM-17 during 5-HT treatment in mesangial cells. IP: immunoprecipitation. Figure shows

    Article Snippet: Cells were cellular proteins were immunoprecipitated using ADAM-17 antibody (QED) or phospho-ERK antibody (Cell Signaling) employing standard immunoprecipiation protocol .

    Techniques: Immunoprecipitation

    Western blot analysis of activation of p38 and JNK and ERK mitogen-activated protein kinases in porcine intestinal epithelial (PIE) cells after challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs). PIE cells were pre-treated with different bacterial strains for 48 h and then stimulated with heat-stable ETEC PAMPs. Phosphorylation of p38, JNK, and ERK was studied at the indicated times post-stimulation. Different letters indicate significant differences (P

    Journal: PLoS ONE

    Article Title: Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

    doi: 10.1371/journal.pone.0059259

    Figure Lengend Snippet: Western blot analysis of activation of p38 and JNK and ERK mitogen-activated protein kinases in porcine intestinal epithelial (PIE) cells after challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs). PIE cells were pre-treated with different bacterial strains for 48 h and then stimulated with heat-stable ETEC PAMPs. Phosphorylation of p38, JNK, and ERK was studied at the indicated times post-stimulation. Different letters indicate significant differences (P

    Article Snippet: Phosphorylation of p38 and JNK and ERK MAPKs, and IκBα degradation were evaluated using anti-phosphorylated p38, anti-phosphorylated JNK, anti-phosphorylated ERK, and anti-IκB antibodies, respectively (Cell Signaling Technology, Beverly, MA), according to the manufacturer’s instructions.

    Techniques: Western Blot, Activation Assay

    Inhibitors for NFκB p65 and ERK 1/2 suppress thrombin-stimulated FN secretion. A : MSCs were pretreated with EP or PD, followed by thrombin treatment with a dose of 4 U/ml. The cells were collected at the indicated time points for Western blotting.  B : The supernatants were collected after MSCs were cultured for 48 hours in the presence or absence of thrombin (TH, 4 U/ml), EP or PD. The concentrations of FN were assessed by ELISA. ** P

    Journal: Stem Cell Research & Therapy

    Article Title: Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways

    doi: 10.1186/scrt424

    Figure Lengend Snippet: Inhibitors for NFκB p65 and ERK 1/2 suppress thrombin-stimulated FN secretion. A : MSCs were pretreated with EP or PD, followed by thrombin treatment with a dose of 4 U/ml. The cells were collected at the indicated time points for Western blotting. B : The supernatants were collected after MSCs were cultured for 48 hours in the presence or absence of thrombin (TH, 4 U/ml), EP or PD. The concentrations of FN were assessed by ELISA. ** P

    Article Snippet: Western blot was performed with the following primary antibodies: rabbit monoclonal antibody anti-phospho-NFκB p65, anti-NFκB p65, anti-phospho-ERK 1/2 and anti-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA, 1:1,000 dilution) in Tris Buffered Saline with Tween (TBST) with 5% bovine serum albumin (BSA).

    Techniques: Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    Thrombin induced the activation of ERK 1/2 and NFκB signalling in MSCs.  MSCs were pre-treated with thrombin (4 U/ml) and collected at the indicated time points for western blotting analysis on the phosphorylation of ERK 1/2 and NFκB p65. The Hela cell line treated with TNF-alpha was used as the positive control. GAPDH was served as the internal reference. The results here are representative of three individual experiments. MSCs, Mesenchymal stem cells; NFκB, Nuclear factor kappa B.

    Journal: Stem Cell Research & Therapy

    Article Title: Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways

    doi: 10.1186/scrt424

    Figure Lengend Snippet: Thrombin induced the activation of ERK 1/2 and NFκB signalling in MSCs. MSCs were pre-treated with thrombin (4 U/ml) and collected at the indicated time points for western blotting analysis on the phosphorylation of ERK 1/2 and NFκB p65. The Hela cell line treated with TNF-alpha was used as the positive control. GAPDH was served as the internal reference. The results here are representative of three individual experiments. MSCs, Mesenchymal stem cells; NFκB, Nuclear factor kappa B.

    Article Snippet: Western blot was performed with the following primary antibodies: rabbit monoclonal antibody anti-phospho-NFκB p65, anti-NFκB p65, anti-phospho-ERK 1/2 and anti-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA, 1:1,000 dilution) in Tris Buffered Saline with Tween (TBST) with 5% bovine serum albumin (BSA).

    Techniques: Activation Assay, Western Blot, Positive Control

    Blockage to PAR affects the phosphorylation of ERK 1/2 and FN secretion by thrombin-treated MSCs.  SCH79797 (SCH) and FSLLRY-NH 2  (FSL) were added into MSC culture in the presence of thrombin (TH, 4 U/ml). The cells were harvested at the indicated time points and the phosphorylated status of ERK 1/2 and NFκB p65 was revealed by Western blotting  (A) . MSC culture was maintained for 48 hours and the supernatants were collected for FN detection by ELISA  (B) . Data are shown as means ± SE (n = 3). *compared with control group (thrombin- and inhibitors- free), * P

    Journal: Stem Cell Research & Therapy

    Article Title: Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways

    doi: 10.1186/scrt424

    Figure Lengend Snippet: Blockage to PAR affects the phosphorylation of ERK 1/2 and FN secretion by thrombin-treated MSCs. SCH79797 (SCH) and FSLLRY-NH 2 (FSL) were added into MSC culture in the presence of thrombin (TH, 4 U/ml). The cells were harvested at the indicated time points and the phosphorylated status of ERK 1/2 and NFκB p65 was revealed by Western blotting (A) . MSC culture was maintained for 48 hours and the supernatants were collected for FN detection by ELISA (B) . Data are shown as means ± SE (n = 3). *compared with control group (thrombin- and inhibitors- free), * P

    Article Snippet: Western blot was performed with the following primary antibodies: rabbit monoclonal antibody anti-phospho-NFκB p65, anti-NFκB p65, anti-phospho-ERK 1/2 and anti-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA, 1:1,000 dilution) in Tris Buffered Saline with Tween (TBST) with 5% bovine serum albumin (BSA).

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

    The key signaling pathways regulating proliferation and metastasis are suppressed during Rhein treatment. Notes: ( A ) 30 or 60 μM Rhein inhibited the expression of p-ERK, p-Akt, MMP9 and CCND1 in A498 and 786-O cells at 48 h. ( B ) The quantitative results of the key molecules changing showed in ( A ) by ImageJ software. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; p-JNK, phospho-c-Jun N-terminal kinase; p-ERK, phospho-extracellular signal-regulated kinase.

    Journal: OncoTargets and therapy

    Article Title: Rhein inhibits malignant phenotypes of human renal cell carcinoma by impacting on MAPK/NF-κB signaling pathways

    doi: 10.2147/OTT.S153798

    Figure Lengend Snippet: The key signaling pathways regulating proliferation and metastasis are suppressed during Rhein treatment. Notes: ( A ) 30 or 60 μM Rhein inhibited the expression of p-ERK, p-Akt, MMP9 and CCND1 in A498 and 786-O cells at 48 h. ( B ) The quantitative results of the key molecules changing showed in ( A ) by ImageJ software. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP9, matrix metalloproteinase 9; p-JNK, phospho-c-Jun N-terminal kinase; p-ERK, phospho-extracellular signal-regulated kinase.

    Article Snippet: Resultant blots were blocked with 5% skim milk and reacted with properly diluted primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz), phospho-extracellular signal-regulated kinase (Santa Cruz), total extracellular signal-regulated kinase (Santa Cruz), p-AKT (Cell Signaling Technology), phospho-c-Jun N-terminal kinase (Cell Signaling Technology), matrix metalloproteinase 9 (MMP9) (Cell Signaling Technology) and CCND1 (Cell Signaling Technology) for 1 h at room temperature.

    Techniques: Expressing, Software

    Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

    Journal: Oncogene

    Article Title: Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML

    doi: 10.1038/onc.2016.41

    Figure Lengend Snippet: Sorafenib-resistant MV4-11 and MOLM-13 cell lines display resistance to multiple FLT3 inhibitors. ( a ) MOLM-13 and MV4-11 cell lines were treated with an increasing concentration (from 0 to 1000 n M ) of multiple tyrosine kinase inhibitors. Cells were cultured with inhibitors for 46 h followed by PrestoBlue viability analysis. ( b ) Sorafenib-sensitive and -resistant cell lines were treated with increasing concentrations of AC220 and sorafenib for 46 h before processing for PrestoBlue viability assays. ( c ) Sorafenib-sensitive and -resistant MOLM-13 and MV4-11 cells were serum-starved for 4 h in the presence or absence of 100 n M sorafenib before 100 ng/ml FL stimulation for 5 min. Cells were then lysed and immunoprecipitated with an anti-FLT3 antibody. The 4G10 (anti-phospho-tyrosine) and anti-FLT3 antibodies were used to probe the blots. ( d ) Cell lysates from the experiment described in c were resolved by SDS–PAGE and analyzed by western blotting using anti-phospho AKT, anti-phospho ERK and anti-Tubulin antibodies. ( e ) MOLM-13 and MV4-11 cells were seeded with or without 100 n M sorafenib in semisolid medium and cultured for 7 days.

    Article Snippet: Additional antibodies include: anti-phosphotyrosine 4G10 (Millipore, Darmstadt, Germany), anti-phospho AKT (Epitomics, Burlingame, CA, USA), anti-phospho ERK (Santa-Cruz Biotechnology Inc., Dallas, TX, USA), anti-phospho S6K and anti-S6K (Abcam, Cambridge, UK), anti-phospho p38 and anti-p38 (BD biosciences, Sparks, MD, USA) and anti-tubulin and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Concentration Assay, Cell Culture, Immunoprecipitation, SDS Page, Western Blot

    The effects of a PI3K/Akt inhibitor and an MEK/ERK inhibitor on tetrandrine (TET)-treated A549 cells. The cells were treated with TET (30 µM) for 24 h in the absence or presence of LY294002 (20 µM) or PD98059 (50 µM). Next, lysates were prepared and Western blot analysis was performed in order to determine protein expression levels.

    Journal: Journal of Veterinary Science

    Article Title: Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells

    doi: 10.4142/jvs.2009.10.1.23

    Figure Lengend Snippet: The effects of a PI3K/Akt inhibitor and an MEK/ERK inhibitor on tetrandrine (TET)-treated A549 cells. The cells were treated with TET (30 µM) for 24 h in the absence or presence of LY294002 (20 µM) or PD98059 (50 µM). Next, lysates were prepared and Western blot analysis was performed in order to determine protein expression levels.

    Article Snippet: Anti-Bid, anti-Bax, anti-Bcl-xL, anti-Akt, anti-phospho-Akt Thr-308, Ser-473, anti-ERK and anti-phospho-ERK antibodies for Western blot analysis were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Western Blot, Expressing

    Incubation of adipose tissue with dexamethasone (0.3 µM) for 24 h or dexamethasone for 24 h together with the CNR1-specific antagonist AM281 (3 µM) for the final 4 h of incubation had no effect on the total protein levels or on the phosphorylation of HSL Ser563 a and ERK Thr202/Tyr204 b ( n = 5). Data are means of densitometry analyses of p-HSL and p-ERK and normalized to the respective total protein levels ( n = 5). GAPDH was used as a loading control protein

    Journal: Endocrine

    Article Title: Role of cannabinoid receptor 1 in human adipose tissue for lipolysis regulation and insulin resistance

    doi: 10.1007/s12020-016-1172-6

    Figure Lengend Snippet: Incubation of adipose tissue with dexamethasone (0.3 µM) for 24 h or dexamethasone for 24 h together with the CNR1-specific antagonist AM281 (3 µM) for the final 4 h of incubation had no effect on the total protein levels or on the phosphorylation of HSL Ser563 a and ERK Thr202/Tyr204 b ( n = 5). Data are means of densitometry analyses of p-HSL and p-ERK and normalized to the respective total protein levels ( n = 5). GAPDH was used as a loading control protein

    Article Snippet: Immunoblotting was performed with equal amount of protein for all samples (10 µg) and with the use of primary antibodies to ERK (4695S, Cell Signaling Technology (CST), Danvers, MA, USA; diluted 1:1000) phospho-ERK (Thr202/Tyr204) (4370S, CST; diluted 1:1000), HSL (4107S, CST; diluted 1:1000) and phospho-HSL (Ser563) (4139S, CST; diluted 1:1000).

    Techniques: Incubation

    DC-ASGPR ligation induces IL-10 from DCs dependent on ERK/p38 contributing to the generation of antigen-specific IL-10–producing CD4 + T cells. (A) IFNDCs (10 5 ) were incubated with indicated inhibitors for 1 h, washed, and loaded with 1 µg/ml IgG4-PSA, α-LOX-1-PSA, or α-DC-ASGPR-PSA. After 24 h, culture supernatants were harvested and IL-10 production was assessed. Error bars indicate the mean ± SEM of two independent experiments with triplicate assay. (B) After 15 min of loading IFNDCs with 1 µg/ml recombinant proteins, α-DC-ASGPR-PSA, or α-LOX-1-PSA, cells were stained with indicated antibodies. Representative data from four independent experiments are presented. (C) 5 × 10 3 IFNDCs were treated with 2.5 µM PD0325901 (MEK inhibitor) or SB203580 (p38 inhibitor) for 1 h and washed thoroughly. DCs were loaded with 1 µg/ml α-DC-ASGPR-PSA or α-LOX-1-PSA. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d. CD4 + T cells were then restimulated with 1 µM PSA 30-44 for 48 h. IL-10, IFN-γ, and IL-2 in culture supernatants were assessed. Each dot represents the data generated with a single experiment. Background values acquired with control peptide (PSA 82-96 ) were substracted. (D) Purified naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with α-DC-ASGPR-PSA–loaded IFNDCs in the presence of control IgG or α–IL-10/IL-10R antibodies for 7 d. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from four independent experiments are presented on the right. (E) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml α-LOX-1-PSA in the presence or absence of 20 pg/ml IL-10. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from five independent experiments are presented in right panel. P-values were calculated with Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells

    doi: 10.1084/jem.20110399

    Figure Lengend Snippet: DC-ASGPR ligation induces IL-10 from DCs dependent on ERK/p38 contributing to the generation of antigen-specific IL-10–producing CD4 + T cells. (A) IFNDCs (10 5 ) were incubated with indicated inhibitors for 1 h, washed, and loaded with 1 µg/ml IgG4-PSA, α-LOX-1-PSA, or α-DC-ASGPR-PSA. After 24 h, culture supernatants were harvested and IL-10 production was assessed. Error bars indicate the mean ± SEM of two independent experiments with triplicate assay. (B) After 15 min of loading IFNDCs with 1 µg/ml recombinant proteins, α-DC-ASGPR-PSA, or α-LOX-1-PSA, cells were stained with indicated antibodies. Representative data from four independent experiments are presented. (C) 5 × 10 3 IFNDCs were treated with 2.5 µM PD0325901 (MEK inhibitor) or SB203580 (p38 inhibitor) for 1 h and washed thoroughly. DCs were loaded with 1 µg/ml α-DC-ASGPR-PSA or α-LOX-1-PSA. CFSE-labeled autologous naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d. CD4 + T cells were then restimulated with 1 µM PSA 30-44 for 48 h. IL-10, IFN-γ, and IL-2 in culture supernatants were assessed. Each dot represents the data generated with a single experiment. Background values acquired with control peptide (PSA 82-96 ) were substracted. (D) Purified naive CD4 + T cells (1–2 × 10 5 ) were co-cultured with α-DC-ASGPR-PSA–loaded IFNDCs in the presence of control IgG or α–IL-10/IL-10R antibodies for 7 d. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from four independent experiments are presented on the right. (E) Naive CD4 + T cells (1–2 × 10 5 ) were co-cultured for 7 d with IFNDCs (5 × 10 3 ) loaded with 1 µg/ml α-LOX-1-PSA in the presence or absence of 20 pg/ml IL-10. Cells were then restimulated with indicated peptides (1 µM) and stained for intracellular IL-10. Summary of the data from five independent experiments are presented in right panel. P-values were calculated with Student’s t test.

    Article Snippet: Cells were stained with primary antibody, rabbit anti-phospho-ERK, or anti-phospho-p38 mitogen-activated protein kinase antibodies (Cell Signaling Technology), and then stained with fluorescently labeled goat anti–rabbit IgG (Sigma-Aldrich).

    Techniques: Ligation, Incubation, Recombinant, Staining, Labeling, Cell Culture, Generated, Purification

    Effect of the calmodulin-dependent PDE inhibitor CGS and CAMKK inhibitor STO on ERK activation. Relative density of pERK/ERK measured by densitometry at 24 h in Saos-2 (A, C) or MG-63 (B, D) cells treated alone with 5 μM CdCl 2 , 5 μM CGS, 5 μM STO, 2.5 μM KN-93 or with CdCl 2 and inhibitor in co-treatment. Controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. Representative Western blots of pERK and ERK for Saos-2 (E, G) and MG-63 (F, H). Each bar represents the mean ± SEM of at least 3 independent experiments. * denotes significant from control p

    Journal: Toxicology letters

    Article Title: Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines

    doi: 10.1016/j.toxlet.2016.08.020

    Figure Lengend Snippet: Effect of the calmodulin-dependent PDE inhibitor CGS and CAMKK inhibitor STO on ERK activation. Relative density of pERK/ERK measured by densitometry at 24 h in Saos-2 (A, C) or MG-63 (B, D) cells treated alone with 5 μM CdCl 2 , 5 μM CGS, 5 μM STO, 2.5 μM KN-93 or with CdCl 2 and inhibitor in co-treatment. Controls received OPTI-MEM serum-free medium containing 0.01% or less DMSO. Representative Western blots of pERK and ERK for Saos-2 (E, G) and MG-63 (F, H). Each bar represents the mean ± SEM of at least 3 independent experiments. * denotes significant from control p

    Article Snippet: Membranes were blocked in TTBS containing 5% nonfat dry milk for 1 h then incubated overnight at 4°C with primary antibodies (Santa Cruz Biotechnology, CA, USA) for phosphorylated ERK (pERK) or total ERK followed by 2 h incubation at room temperature with HRP antibody (Santa Cruz Biotechnology, CA, USA).

    Techniques: Activation Assay, Western Blot

    (a) The representative protein product of p-EKR extracted from the left ventricles of excised hearts in hamsters of control, cholesterol and Li-Fu formula groups were measured by western blotting analysis. (b) Bars represent the relative protein quantification of p-ERK on the basis of α -tubulin. All bars indicate mean values ± SD ( n = 6 in each group). ** P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Ameliorate Effects of Li-Fu Formula on IL-6-Mediated Cardiac Hypertrophy in Hamsters Fed with a Hyper-Cholesterol Diet

    doi: 10.1093/ecam/neq066

    Figure Lengend Snippet: (a) The representative protein product of p-EKR extracted from the left ventricles of excised hearts in hamsters of control, cholesterol and Li-Fu formula groups were measured by western blotting analysis. (b) Bars represent the relative protein quantification of p-ERK on the basis of α -tubulin. All bars indicate mean values ± SD ( n = 6 in each group). ** P

    Article Snippet: After blocking with 5% non-fat dry milk in PBS for 30 min at room temperature, antibodies against ANP, BNP, IL-6, STAT3, MEK5, p-EKR5, MEK, p-MEK, phosphorylated ERK (p-ERK), p-P38, JNK, p-JNK and α -tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted to 1:500 with 5% nonfat dry milk in PBS and then incubated for 1.5 h at room temperature.

    Techniques: Western Blot

    Our proposed hypothesis that cardiac IL-6, MEK-5-ERK-5 and STAT3 hypertrophic pathways and MEK1/2-ERK1/2 non-cardiacmyocyte proliferative pathway are more activated in hyper cholesterol-fed hamster hearts. The eccentric hypertrophy-related pathway, IL-6-related MEK5-ERK5 pathways and MEK1/2-ERK1/2 non-cardiacmyocyte proliferative pathway may play a part of role for developing eccentric cardiac hypertrophy and pathological changes in hyper cholesterol-fed hamster hearts. Dash lines represent possible theoretical pathways but are still unconfirmed. Up arrows and down arrows on the right side represent increases and decreases, respectively.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Ameliorate Effects of Li-Fu Formula on IL-6-Mediated Cardiac Hypertrophy in Hamsters Fed with a Hyper-Cholesterol Diet

    doi: 10.1093/ecam/neq066

    Figure Lengend Snippet: Our proposed hypothesis that cardiac IL-6, MEK-5-ERK-5 and STAT3 hypertrophic pathways and MEK1/2-ERK1/2 non-cardiacmyocyte proliferative pathway are more activated in hyper cholesterol-fed hamster hearts. The eccentric hypertrophy-related pathway, IL-6-related MEK5-ERK5 pathways and MEK1/2-ERK1/2 non-cardiacmyocyte proliferative pathway may play a part of role for developing eccentric cardiac hypertrophy and pathological changes in hyper cholesterol-fed hamster hearts. Dash lines represent possible theoretical pathways but are still unconfirmed. Up arrows and down arrows on the right side represent increases and decreases, respectively.

    Article Snippet: After blocking with 5% non-fat dry milk in PBS for 30 min at room temperature, antibodies against ANP, BNP, IL-6, STAT3, MEK5, p-EKR5, MEK, p-MEK, phosphorylated ERK (p-ERK), p-P38, JNK, p-JNK and α -tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted to 1:500 with 5% nonfat dry milk in PBS and then incubated for 1.5 h at room temperature.

    Techniques:

    Modulation of T Cell Activation by DNA-CARζ Length and Sequence . (B) Dose-response curves for DNA ligands of varying hybridization lengths. The total ligand DNA length remained constant by adding non-hybridizing Ts. Mean ± SD (n = 3). (C) Stepwise conversion of A/T to G/C base pairs increases the potency of the 11-mer DNA ligand for inducing phosphoERK signaling. Scale bar, 100 μm. Mean ± SD of each ligand density measured in triplicate from one representative experiment. (D) Single ligand-receptor lifetimes by TIRF microscopy. An example distribution of lifetimes from one DNA ligand. The observed (τ obs ) and photobleach-corrected (τ corr .

    Journal: Cell

    Article Title: A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination

    doi: 10.1016/j.cell.2017.03.006

    Figure Lengend Snippet: Modulation of T Cell Activation by DNA-CARζ Length and Sequence . (B) Dose-response curves for DNA ligands of varying hybridization lengths. The total ligand DNA length remained constant by adding non-hybridizing Ts. Mean ± SD (n = 3). (C) Stepwise conversion of A/T to G/C base pairs increases the potency of the 11-mer DNA ligand for inducing phosphoERK signaling. Scale bar, 100 μm. Mean ± SD of each ligand density measured in triplicate from one representative experiment. (D) Single ligand-receptor lifetimes by TIRF microscopy. An example distribution of lifetimes from one DNA ligand. The observed (τ obs ) and photobleach-corrected (τ corr .

    Article Snippet: Cells were labeled over night with anti-phosphoERK (rabbit polyclonal, Cell Signaling Technology #9101, used at 1:500).

    Techniques: Activation Assay, Sequencing, Hybridization, Microscopy