phospho-erk Search Results


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  • 92
    Cell Signaling Technology Inc phosphorylated extracellular signal regulated kinase phospho erk
    Induction of phosphorylated <t>ERK</t> 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 <t>ERK1/2</t> 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
    Phosphorylated Extracellular Signal Regulated Kinase Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc phospho extracellular signal regulated kinase phospho erk antibodies
    Induction of phosphorylated <t>ERK</t> 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 <t>ERK1/2</t> 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
    Phospho Extracellular Signal Regulated Kinase Phospho Erk Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc rabbit phosphorylated extracellular signal regulated kinase phospho erk
    Induction of phosphorylated <t>ERK</t> 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 <t>ERK1/2</t> 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
    Rabbit Phosphorylated Extracellular Signal Regulated Kinase Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam phosphorylated extracellular signal regulated kinase p erk
    Induction of phosphorylated <t>ERK</t> 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 <t>ERK1/2</t> 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
    Phosphorylated Extracellular Signal Regulated Kinase P Erk, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti phosphor extracellular signal regulated kinase p erk
    Induction of phosphorylated <t>ERK</t> 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 <t>ERK1/2</t> 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
    Anti Phosphor Extracellular Signal Regulated Kinase P Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology phosphorylated erk
    Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a <t>Phosphorylated-ERK,</t> phosphorylated-JNK, <t>phosphorylated-p38,</t> or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis
    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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    93
    Thermo Fisher phosphorylation phospho erk
    Knockdown of <t>ANO6</t> inhibited the activation of <t>ERK</t> signaling. After T98G and U87MG cells transfection with ANO6-KD 1 or ANO6-KD2, ( A and B ) the protein expression level of ERK and phospho-ERK was detected by Western blot assay; ( C ) the protein expression levels of ANO6 and ERK were detected by Western blot assay in cytoplasm and cell nucleus of T98G cells; ( D and E ) Histograms were used to represent statistical results of the protein expression levels of ANO6 and ERK in cytoplasm and cell nucleus of T98G cells; ( F ) the protein expression levels of ANO6 and ERK were detected by Western blot assay in cytoplasm and cell nucleus of U87MG cells. ( G and H ) Histograms were used to represent statistical results of the protein expression levels of ANO6 and ERK in cytoplasm and cell nucleus of U87MG cells; β-actin was used as a load control for cytoplasm. LaminB was used as a load control for cell nucleus. Data are presented as the mean ± standard deviation. ** p
    Phosphorylation Phospho Erk, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson phospho erk
    SKAP-55 deficient T-cells show enhanced anti-CD3 induced <t>ERK</t> activation. Panel A: <t>FACS</t> profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .
    Phospho Erk, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 112 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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    88
    Millipore phospho extracellular signal regulated kinase erk
    Increased PDGFRα and -β protein is associated with activated <t>ERK-1/-2</t> and Akt. Protein lysates were made from livers of Tg and WT mice at the indicated ages, and immunoblot analyses were performed as described in Materials and Methods . ( A ). Livers from Tg ( n = 3) and WT ( n = 4) mice at 4 weeks of age contain elevated levels of PDGFRα and -β. A nonspecific band (n.s.) was used as loading control. ( B ). Elevated levels of PDGFRβ protein are detected at all time points ( Top ). Phosphospecific antibodies detected active (phosphorylated) ERK-1/-2 ( Middle ) and Akt ( Bottom ) in the lysates of PDGF-C Tg mice but not in WT mice. Total levels of ERK-1/-2 and Akt are shown.
    Phospho Extracellular Signal Regulated Kinase Erk, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ECM Biosciences phospho erk
    Increased PDGFRα and -β protein is associated with activated <t>ERK-1/-2</t> and Akt. Protein lysates were made from livers of Tg and WT mice at the indicated ages, and immunoblot analyses were performed as described in Materials and Methods . ( A ). Livers from Tg ( n = 3) and WT ( n = 4) mice at 4 weeks of age contain elevated levels of PDGFRα and -β. A nonspecific band (n.s.) was used as loading control. ( B ). Elevated levels of PDGFRβ protein are detected at all time points ( Top ). Phosphospecific antibodies detected active (phosphorylated) ERK-1/-2 ( Middle ) and Akt ( Bottom ) in the lysates of PDGF-C Tg mice but not in WT mice. Total levels of ERK-1/-2 and Akt are shown.
    Phospho Erk, supplied by ECM Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phospho erk
    Activation of MAP kinase and NF-κB upon injury of murine cartilage. Three murine hip explants were avulsed onto dry ice (time 0) or into serum-free medium for up to 90 minutes. For some experiments, interleukin-1 (IL-1) was used as a positive control. A, Lysates were prepared and then Western blotted for phosphorylated <t>ERK,</t> <t>p38,</t> and JNK, as well as total ERK (loading control). B, Some explants were snap frozen, embedded in OCT, sectioned, and the sections were stained for visualization of p65 (a component of the NF-κB pathway) (green) and propidium iodide (for nuclei) (red). Signal was detected with fluorescence-tagged secondary antibodies and visualized with confocal microscopy. Also shown are Safranin O–stained sections taken from adjacent regions of the tissue. C, Three separate gradient images were taken from 3 different regions of the explant (superficial zone, middle zone, and epiphyseal line). The percentage of cells demonstrating translocation of p65 (nuclear signal changing from red to yellow) was expressed as a percentage of the total number of nuclei. Values are the mean ± SD. P
    Phospho Erk, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cisbio Bioassays phospho erk
    Activation of MAP kinase and NF-κB upon injury of murine cartilage. Three murine hip explants were avulsed onto dry ice (time 0) or into serum-free medium for up to 90 minutes. For some experiments, interleukin-1 (IL-1) was used as a positive control. A, Lysates were prepared and then Western blotted for phosphorylated <t>ERK,</t> <t>p38,</t> and JNK, as well as total ERK (loading control). B, Some explants were snap frozen, embedded in OCT, sectioned, and the sections were stained for visualization of p65 (a component of the NF-κB pathway) (green) and propidium iodide (for nuclei) (red). Signal was detected with fluorescence-tagged secondary antibodies and visualized with confocal microscopy. Also shown are Safranin O–stained sections taken from adjacent regions of the tissue. C, Three separate gradient images were taken from 3 different regions of the explant (superficial zone, middle zone, and epiphyseal line). The percentage of cells demonstrating translocation of p65 (nuclear signal changing from red to yellow) was expressed as a percentage of the total number of nuclei. Values are the mean ± SD. P
    Phospho Erk, supplied by Cisbio Bioassays, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho erk/product/Cisbio Bioassays
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    Image Search Results


    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

    Luteolin and luteolin-7- O -glucoside inhibited phosphorylation of Akt in LPS-stimulated RAW 264.7 cells. Panel A shows protein expression levels of p-Akt, p-ERK, p-JNK and p-p38 in response to luteolin and luteolin-7- O -glucoside. All signals were normalized to protein levels of Akt, ERK, JNK and p38 internal controls, an expressed as a ratio (Panel B). Data represent the mean ± SD of triplicate experiments. Values sharing the same superscript are not significantly different at P

    Journal: Nutrition Research and Practice

    Article Title: Luteolin and luteolin-7-O-glucoside inhibit lipopolysaccharide-induced inflammatory responses through modulation of NF-?B/AP-1/PI3K-Akt signaling cascades in RAW 264.7 cells

    doi: 10.4162/nrp.2013.7.6.423

    Figure Lengend Snippet: Luteolin and luteolin-7- O -glucoside inhibited phosphorylation of Akt in LPS-stimulated RAW 264.7 cells. Panel A shows protein expression levels of p-Akt, p-ERK, p-JNK and p-p38 in response to luteolin and luteolin-7- O -glucoside. All signals were normalized to protein levels of Akt, ERK, JNK and p38 internal controls, an expressed as a ratio (Panel B). Data represent the mean ± SD of triplicate experiments. Values sharing the same superscript are not significantly different at P

    Article Snippet: Antibodies against iNOS, COX-2, phospho-p65, p65, phospho-c-jun, c-jun, phospho-extracellular signal-regulated kinase (ERK), ERK, phosphor-c-Jun NH2 -terminal kinase (JNK), JNK, phospho-p38, p38, phosphor-Akt, Akt, and β-actin as well as horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were purchased from Cell Signaling (Boston, MA, USA).

    Techniques: Expressing

    Effects of NTCU and LPS, alone and in combination, on inflammation-related proteins and modulation of these effects by dietary DIM. A, Mouse lung tissue levels of NF-κB, STAT3, p-38 and ERK activation and expression of Mcl-1 and COX-2, downstream effectors of NF-κB and STAT3, respectively, were determined by Western immunoblotting as described in the materials and methods section. B, quantification of the western blot results. Densitometry measurements of Western blot bands were performed using digitalized scientific software program UN-SCAN-IT software. Effects of NTCU and/or LPS on NF-κB-DNA binding (C) and STAT3-DNA binding (D) tissues as determined by ELISA-based EMSA assays (Active motif). Values are presented as mean ± SD. *, P

    Journal: Cancer prevention research (Philadelphia, Pa.)

    Article Title: Dietary diindolylmethane suppresses inflammation-driven lung squamous cell carcinoma in mice

    doi: 10.1158/1940-6207.CAPR-14-0245

    Figure Lengend Snippet: Effects of NTCU and LPS, alone and in combination, on inflammation-related proteins and modulation of these effects by dietary DIM. A, Mouse lung tissue levels of NF-κB, STAT3, p-38 and ERK activation and expression of Mcl-1 and COX-2, downstream effectors of NF-κB and STAT3, respectively, were determined by Western immunoblotting as described in the materials and methods section. B, quantification of the western blot results. Densitometry measurements of Western blot bands were performed using digitalized scientific software program UN-SCAN-IT software. Effects of NTCU and/or LPS on NF-κB-DNA binding (C) and STAT3-DNA binding (D) tissues as determined by ELISA-based EMSA assays (Active motif). Values are presented as mean ± SD. *, P

    Article Snippet: Anti-phospho-STAT3, anti-total STAT3, anti-phospho-Akt, anti-total Akt, anti-phospho-extracellular signal-regulated kinase (ERK), anti-total ERK, anti-phospho-p38, total p-38, anti-Mcl-1, anti-p53, anti-COX2, anti-phospho IκBα, anti-total IκBα, anti-Bax, anti-p-21, anti-PARP, anti-β-actin and goat anti-rabbit IgG secondary antibody were from Cell Signaling Technology (Beverly, MA).

    Techniques: Activation Assay, Expressing, Western Blot, Software, Binding Assay, Enzyme-linked Immunosorbent Assay

    Prevention of chemerin-induced insulin resistance by ERK inhibition. A : Skeletal muscle cells from different donors were precultured with or without 50 μmol/l of the specific ERK inhibitor PD 98059 for 15 min before starting the administration with chemerin or TNF-α. The cells were then treated with chemerin for 30 min and as a control with 2.5 nmol/l TNF-α for 10 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for ERK-1/2 and tubulin for loading control. Representative blots are shown. B and C : After pretreatment for 15 min with PD 98059 (50 μmol/l), skeletal muscle cells from different donors were treated with chemerin overnight. After insulin stimulation, total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for Akt and tubulin for loading control. Representative blots are shown. Data are the means ± SEM of four independent experiments. Glucose uptake was measured as outlined in the research design and methods section. Data are the means ± SEM of three independent experiments. *Significantly different from respective insulin-stimulated control. ■, insulin; □, basal.

    Journal: Diabetes

    Article Title: Chemerin Is a Novel Adipocyte-Derived Factor Inducing Insulin Resistance in Primary Human Skeletal Muscle Cells

    doi: 10.2337/db09-0277

    Figure Lengend Snippet: Prevention of chemerin-induced insulin resistance by ERK inhibition. A : Skeletal muscle cells from different donors were precultured with or without 50 μmol/l of the specific ERK inhibitor PD 98059 for 15 min before starting the administration with chemerin or TNF-α. The cells were then treated with chemerin for 30 min and as a control with 2.5 nmol/l TNF-α for 10 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for ERK-1/2 and tubulin for loading control. Representative blots are shown. B and C : After pretreatment for 15 min with PD 98059 (50 μmol/l), skeletal muscle cells from different donors were treated with chemerin overnight. After insulin stimulation, total cell lysates were resolved by SDS-PAGE and immunoblotted with a phosphospecific antibody for Akt and tubulin for loading control. Representative blots are shown. Data are the means ± SEM of four independent experiments. Glucose uptake was measured as outlined in the research design and methods section. Data are the means ± SEM of three independent experiments. *Significantly different from respective insulin-stimulated control. ■, insulin; □, basal.

    Article Snippet: Polyclonal antibodies anti–phospho-glycogen synthase kinase (phospho-GSK)3α/β (Ser21/9), anti–phospho-Akt (Ser473), anti–phospho-nuclear factor-κB (NF-κB [p65, Ser536]), anti–phospho-extracellular signal–regulated kinase (phospho-ERK)-1/2 (Thr202/Tyr204), and anti–phospho-p38 mitogen-activated protein (phospho-p38 MAP) kinase (Thr180/Tyr182) were supplied by Cell Signaling Technology (Frankfurt, Germany) and anti-tubulin from Calbiochem (Merck Biosciences, Schwalbach, Germany).

    Techniques: Inhibition, SDS Page

    Chemerin signaling in skeletal muscle cells. A : Skeletal muscle cells from different donors were cultured with chemerin for 30 min and as a control with 2.5 nmol/l TNF-α for 10 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Representative blots are shown. B : Skeletal muscle cells from different donors were cultured with chemerin for 10–120 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Data are the means ± SEM of four to five independent experiments. *Significantly different from unstimulated control. C : Skeletal muscle cells from different donors were cultured with different concentrations of chemerin for 24 h. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Data are the means ± SEM of four to five independent experiments. *Significantly different from unstimulated control.

    Journal: Diabetes

    Article Title: Chemerin Is a Novel Adipocyte-Derived Factor Inducing Insulin Resistance in Primary Human Skeletal Muscle Cells

    doi: 10.2337/db09-0277

    Figure Lengend Snippet: Chemerin signaling in skeletal muscle cells. A : Skeletal muscle cells from different donors were cultured with chemerin for 30 min and as a control with 2.5 nmol/l TNF-α for 10 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Representative blots are shown. B : Skeletal muscle cells from different donors were cultured with chemerin for 10–120 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Data are the means ± SEM of four to five independent experiments. *Significantly different from unstimulated control. C : Skeletal muscle cells from different donors were cultured with different concentrations of chemerin for 24 h. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phosphospecific antibodies for p38 MAP kinase, the p65 subunit of NF-κB (p65), and ERK-1/2 and tubulin for loading control. Data are the means ± SEM of four to five independent experiments. *Significantly different from unstimulated control.

    Article Snippet: Polyclonal antibodies anti–phospho-glycogen synthase kinase (phospho-GSK)3α/β (Ser21/9), anti–phospho-Akt (Ser473), anti–phospho-nuclear factor-κB (NF-κB [p65, Ser536]), anti–phospho-extracellular signal–regulated kinase (phospho-ERK)-1/2 (Thr202/Tyr204), and anti–phospho-p38 mitogen-activated protein (phospho-p38 MAP) kinase (Thr180/Tyr182) were supplied by Cell Signaling Technology (Frankfurt, Germany) and anti-tubulin from Calbiochem (Merck Biosciences, Schwalbach, Germany).

    Techniques: Cell Culture, SDS Page

    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

    The ERK/MAPK signaling pathway was regulated when THP-1-derived macrophages were treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours. Notes: The proteomic response was determined by SILAC, and pathways were analyzed using IPA. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red colors indicates the degree of down- or upregulation. Solid arrow indicates direct interaction, and dashed arrow indicates indirect interaction. Abbreviations: ERK, extracellular signal-regulated kinase; IPA, ingenuity pathway analysis; MAPK, mitogen-activated protein kinase; NP, nanoparticle; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Novel targeting of PEGylated liposomes for codelivery of TGF-β1 siRNA and four antitubercular drugs to human macrophages for the treatment of mycobacterial infection: a quantitative proteomic study

    doi: 10.2147/DDDT.S79369

    Figure Lengend Snippet: The ERK/MAPK signaling pathway was regulated when THP-1-derived macrophages were treated with the novel NP-siRNA liposomes at 12.5 μg/mL for 24 hours. Notes: The proteomic response was determined by SILAC, and pathways were analyzed using IPA. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red colors indicates the degree of down- or upregulation. Solid arrow indicates direct interaction, and dashed arrow indicates indirect interaction. Abbreviations: ERK, extracellular signal-regulated kinase; IPA, ingenuity pathway analysis; MAPK, mitogen-activated protein kinase; NP, nanoparticle; SILAC, stable isotope labeling with amino acids in cell culture; siRNA, small interfering RNA.

    Article Snippet: Primary antibodies against human phosphorylated phosphoinositide 3-kinase (p-PI3K), phosphorylated p38 mitogenactivated protein kinase (p-p38 MAPK), p38 MAPK, mammalian target of rapamycin (mTOR), p-mTOR, phosphorylated extracellular signal-regulated kinase (ERK), p-ERK, phosphatase and tensin homologue (PTEN), beclin 1, microtubule-associated protein 1A/1B-light chain 3 (LC3), nuclear factor-κB (NF-κB), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

    Techniques: Derivative Assay, Indirect Immunoperoxidase Assay, Labeling, Cell Culture, Small Interfering RNA

    Effect of apigenin on the TGF-β1-induced MAPK signaling pathway in nasal fibroblasts. Nasal fibroblasts were pretreated with apigenin (5 μM) for 1 hour and combined with TGF-β1 (5 ng/ml). (A) Phosphorylation of MAPK (p-p38, p-JNK, and p-ERK) was detected by western blot. (B) Nasal fibroblasts were stimulated with TGF-β1 with or without the following specific inhibitors: SB203580 (10 μM), SP600125 (5 μM). Protein expression levels of a -SMA, fibronectin, and collagen type I were determined by western blot. (C) Total collagen was evaluated by sircol assay. Results were obtained from at least three independent experiments. * p

    Journal: PLoS ONE

    Article Title: Apigenin alleviates TGF-β1-induced nasal mucosa remodeling by inhibiting MAPK / NF-kB signaling pathways in chronic rhinosinusitis

    doi: 10.1371/journal.pone.0201595

    Figure Lengend Snippet: Effect of apigenin on the TGF-β1-induced MAPK signaling pathway in nasal fibroblasts. Nasal fibroblasts were pretreated with apigenin (5 μM) for 1 hour and combined with TGF-β1 (5 ng/ml). (A) Phosphorylation of MAPK (p-p38, p-JNK, and p-ERK) was detected by western blot. (B) Nasal fibroblasts were stimulated with TGF-β1 with or without the following specific inhibitors: SB203580 (10 μM), SP600125 (5 μM). Protein expression levels of a -SMA, fibronectin, and collagen type I were determined by western blot. (C) Total collagen was evaluated by sircol assay. Results were obtained from at least three independent experiments. * p

    Article Snippet: The blots were incubated with primary antibodies against α-SMA (Millipore Inc.), fibronectin ( Santa Cruz Biotecknology Inc., CA ), collagen type I (Abcam, Cambridge, UK), phospho-p38, total-p38, phospho-JNK, total-JNK, phospho-ERK, total-ERK (Cell Signaling, MA), total-p50, phospho-p50 (Santa Cruz Biotecknology Inc.), and β-actin (Santa Cruz Biotecknology Inc.) overnight at 4°C.

    Techniques: Western Blot, Expressing

    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

    Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

    Journal: BMC Complementary and Alternative Medicine

    Article Title: 4-Acetylantroquinonol B inhibits lipopolysaccharide-induced cytokine release and alleviates sepsis through of MAPK and NFκB suppression

    doi: 10.1186/s12906-018-2172-2

    Figure Lengend Snippet: Effects of 4AAQB on the LPS-induced activation of MAP kinases, IkBα, NFκB p65 and STAT1 in RAW 264.7 macrophages and peritoneal macrophages. RAW264.7 cells were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Cells were harvested and total cell extracts were prepared. a Phosphorylated-ERK, phosphorylated-JNK, phosphorylated-p38, or b Phosphorylated-IκBα and NFκB p65 subunit and c Phosphorylated-STAT1 were detected by Western blot analysis. Total ERK, JNK, p38 and α-tubulin were used as internal standard. d Peritoneal macrophages were treated with various concentration of 4AAQB and stimulated with LPS (100 ng/ml) for 30 min. Phosphorylated-ERK and total ERK were detected by Western blot analysis

    Article Snippet: Antibodies against p38, phosphorylated p38, JNK, phosphorylated JNK, ERK, phosphorylated ERK, α-tubulin, iNOS, phosphorylated NFκB p65 were purchased from Santa Cruz (Biotechnology, Inc., USA). β-actin antibody was from Novus Biologicals (Littleton, CO, USA).

    Techniques: Activation Assay, Concentration 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:

    Knockdown of ANO6 inhibited the activation of ERK signaling. After T98G and U87MG cells transfection with ANO6-KD 1 or ANO6-KD2, ( A and B ) the protein expression level of ERK and phospho-ERK was detected by Western blot assay; ( C ) the protein expression levels of ANO6 and ERK were detected by Western blot assay in cytoplasm and cell nucleus of T98G cells; ( D and E ) Histograms were used to represent statistical results of the protein expression levels of ANO6 and ERK in cytoplasm and cell nucleus of T98G cells; ( F ) the protein expression levels of ANO6 and ERK were detected by Western blot assay in cytoplasm and cell nucleus of U87MG cells. ( G and H ) Histograms were used to represent statistical results of the protein expression levels of ANO6 and ERK in cytoplasm and cell nucleus of U87MG cells; β-actin was used as a load control for cytoplasm. LaminB was used as a load control for cell nucleus. Data are presented as the mean ± standard deviation. ** p

    Journal: OncoTargets and therapy

    Article Title: ANO6 promotes cell proliferation and invasion in glioma through regulating the ERK signaling pathway

    doi: 10.2147/OTT.S211725

    Figure Lengend Snippet: Knockdown of ANO6 inhibited the activation of ERK signaling. After T98G and U87MG cells transfection with ANO6-KD 1 or ANO6-KD2, ( A and B ) the protein expression level of ERK and phospho-ERK was detected by Western blot assay; ( C ) the protein expression levels of ANO6 and ERK were detected by Western blot assay in cytoplasm and cell nucleus of T98G cells; ( D and E ) Histograms were used to represent statistical results of the protein expression levels of ANO6 and ERK in cytoplasm and cell nucleus of T98G cells; ( F ) the protein expression levels of ANO6 and ERK were detected by Western blot assay in cytoplasm and cell nucleus of U87MG cells. ( G and H ) Histograms were used to represent statistical results of the protein expression levels of ANO6 and ERK in cytoplasm and cell nucleus of U87MG cells; β-actin was used as a load control for cytoplasm. LaminB was used as a load control for cell nucleus. Data are presented as the mean ± standard deviation. ** p

    Article Snippet: The antibodies are as follows: ANO6 (1:1000, cat#PA5-69345, ThermoFisher, California, USA), ERK (1:1000, cat#13–6200, ThermoFisher, California, USA), phosphorylation (phospho)-ERK (1:500, cat#44-680G, ThermoFisher, California, USA), LaminB (1:2000, cat#PA5-66473, ThermoFisher, California, USA), β-actin (1:5000, cat#MA5-15739, ThermoFisher, California, USA).

    Techniques: Activation Assay, Transfection, Expressing, Western Blot, Standard Deviation

    ERK inhibitor inhibited the proliferation and invasion of ANO6 overexpression cells. After SHG-44 cells transfection with ANO6 plasmid was treated with PD98059, ( A ) the cell viability was detected by MTT assay; ( B and C ) the ability of cell proliferation was detected by colony formation; ( D and E ) the ability of invasion was detected by transwell assay. Data are presented as the mean ± standard deviation. ** p

    Journal: OncoTargets and therapy

    Article Title: ANO6 promotes cell proliferation and invasion in glioma through regulating the ERK signaling pathway

    doi: 10.2147/OTT.S211725

    Figure Lengend Snippet: ERK inhibitor inhibited the proliferation and invasion of ANO6 overexpression cells. After SHG-44 cells transfection with ANO6 plasmid was treated with PD98059, ( A ) the cell viability was detected by MTT assay; ( B and C ) the ability of cell proliferation was detected by colony formation; ( D and E ) the ability of invasion was detected by transwell assay. Data are presented as the mean ± standard deviation. ** p

    Article Snippet: The antibodies are as follows: ANO6 (1:1000, cat#PA5-69345, ThermoFisher, California, USA), ERK (1:1000, cat#13–6200, ThermoFisher, California, USA), phosphorylation (phospho)-ERK (1:500, cat#44-680G, ThermoFisher, California, USA), LaminB (1:2000, cat#PA5-66473, ThermoFisher, California, USA), β-actin (1:5000, cat#MA5-15739, ThermoFisher, California, USA).

    Techniques: Over Expression, Transfection, Plasmid Preparation, MTT Assay, Transwell Assay, Standard Deviation

    SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation. Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .

    Journal: PLoS ONE

    Article Title: Adaptor SKAP-55 Binds p21ras Activating Exchange Factor RasGRP1 and Negatively Regulates the p21ras-ERK Pathway in T-Cells

    doi: 10.1371/journal.pone.0001718

    Figure Lengend Snippet: SKAP-55 deficient T-cells show enhanced anti-CD3 induced ERK activation. Panel A: FACS profile of T-cells from SKAP-55+/+, +/− and −/− mice stained with AlexaFluor647 labeled anti-pERK. T-cells from lymph-nodes were left unstimulated, or stimulated with anti-CD3-biotin (10 µg/ml) and streptavidin for 5 min. Upper panel: SKAP-55+/+; upper middle panel: SKAP-55−/+; lower middle panel: SKAP-55−/−; lower panel: comparison of anti-CD3 stimulated SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells vs. pervanadate treated cells. Right upper panel: histogram showing the difference in MFI values for SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells. Right lower panel: histogram showing the difference in the percentage of SKAP-55+/+, SKAP-55+/− and SKAP-55−/− cells staining for pERK. Panel B: Anti-pERK immunoblotting of anti-CD3 activated SKAP-55+/+ versus SKAP-55−/− T-cells. T-cells were activated with 5 µg/ml anti-CD3 for 1–30 minutes. Upper panel: anti-pERK blot; middle panel: anti-ERK blotting; lower panel: anti-SKAP-55 blot. SKAP-55+/+: lanes 1–7; SKAP-55−/−: lanes 8–14; Resting: lanes 1, 8; Stimulated: 1 min: lanes 2,9; 2 min: lanes 3, 10; 5 min: lanes 4,11; 10 min: lanes 5,12; 15 min: lanes 6, 13; 30 min: lane 7, 14. Panel C: SKAP-55 deficient T-cells have impaired adhesion to ICAM-1. Cells were stimulated with anti-CD3 followed by a measurement of binding to immobilized ICAM-1 on plates as described in Material and Methods .

    Article Snippet: FACS staining and Immunofluorescence For detection of phospho-ERK by flow cytometry, cells were permeabilized, stained with AlexaFluor647 tagged anti-phospho-ERK1/2 and analyzed by FACS (BD FacsCalibur), as described .

    Techniques: Activation Assay, FACS, Mouse Assay, Staining, Labeling, Binding 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

    Increased PDGFRα and -β protein is associated with activated ERK-1/-2 and Akt. Protein lysates were made from livers of Tg and WT mice at the indicated ages, and immunoblot analyses were performed as described in Materials and Methods . ( A ). Livers from Tg ( n = 3) and WT ( n = 4) mice at 4 weeks of age contain elevated levels of PDGFRα and -β. A nonspecific band (n.s.) was used as loading control. ( B ). Elevated levels of PDGFRβ protein are detected at all time points ( Top ). Phosphospecific antibodies detected active (phosphorylated) ERK-1/-2 ( Middle ) and Akt ( Bottom ) in the lysates of PDGF-C Tg mice but not in WT mice. Total levels of ERK-1/-2 and Akt are shown.

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

    Article Title: Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma

    doi: 10.1073/pnas.0409722102

    Figure Lengend Snippet: Increased PDGFRα and -β protein is associated with activated ERK-1/-2 and Akt. Protein lysates were made from livers of Tg and WT mice at the indicated ages, and immunoblot analyses were performed as described in Materials and Methods . ( A ). Livers from Tg ( n = 3) and WT ( n = 4) mice at 4 weeks of age contain elevated levels of PDGFRα and -β. A nonspecific band (n.s.) was used as loading control. ( B ). Elevated levels of PDGFRβ protein are detected at all time points ( Top ). Phosphospecific antibodies detected active (phosphorylated) ERK-1/-2 ( Middle ) and Akt ( Bottom ) in the lysates of PDGF-C Tg mice but not in WT mice. Total levels of ERK-1/-2 and Akt are shown.

    Article Snippet: Protein levels were determined with specific antibodies to PDGFRα (Lab Vision, Fremont, CA), PDGFRβ (Upstate Biotechnology), phospho-extracellular signal-regulated kinase (ERK)-1/-2 (Sigma), and phospho-Akt (Ser-473 and Thr-308) and Akt (Cell Signaling Technology, Beverly, MA).

    Techniques: Mouse Assay

    Activation of MAP kinase and NF-κB upon injury of murine cartilage. Three murine hip explants were avulsed onto dry ice (time 0) or into serum-free medium for up to 90 minutes. For some experiments, interleukin-1 (IL-1) was used as a positive control. A, Lysates were prepared and then Western blotted for phosphorylated ERK, p38, and JNK, as well as total ERK (loading control). B, Some explants were snap frozen, embedded in OCT, sectioned, and the sections were stained for visualization of p65 (a component of the NF-κB pathway) (green) and propidium iodide (for nuclei) (red). Signal was detected with fluorescence-tagged secondary antibodies and visualized with confocal microscopy. Also shown are Safranin O–stained sections taken from adjacent regions of the tissue. C, Three separate gradient images were taken from 3 different regions of the explant (superficial zone, middle zone, and epiphyseal line). The percentage of cells demonstrating translocation of p65 (nuclear signal changing from red to yellow) was expressed as a percentage of the total number of nuclei. Values are the mean ± SD. P

    Journal: Arthritis and Rheumatism

    Article Title: Fibroblast Growth Factor 2 Drives Changes in Gene Expression Following Injury to Murine Cartilage In Vitro and In Vivo

    doi: 10.1002/art.38039

    Figure Lengend Snippet: Activation of MAP kinase and NF-κB upon injury of murine cartilage. Three murine hip explants were avulsed onto dry ice (time 0) or into serum-free medium for up to 90 minutes. For some experiments, interleukin-1 (IL-1) was used as a positive control. A, Lysates were prepared and then Western blotted for phosphorylated ERK, p38, and JNK, as well as total ERK (loading control). B, Some explants were snap frozen, embedded in OCT, sectioned, and the sections were stained for visualization of p65 (a component of the NF-κB pathway) (green) and propidium iodide (for nuclei) (red). Signal was detected with fluorescence-tagged secondary antibodies and visualized with confocal microscopy. Also shown are Safranin O–stained sections taken from adjacent regions of the tissue. C, Three separate gradient images were taken from 3 different regions of the explant (superficial zone, middle zone, and epiphyseal line). The percentage of cells demonstrating translocation of p65 (nuclear signal changing from red to yellow) was expressed as a percentage of the total number of nuclei. Values are the mean ± SD. P

    Article Snippet: Transferred membranes were Western blotted for phospho-JNK (1:1,000 dilution; Cell Signaling Technology catalog no. 9251), phospho-p38 (1:1,000 dilution; Cell Signaling Technology catalog no. 9211), phospho-ERK (1:1,000 dilution; Sigma-Aldrich catalog no. 8159), total ERK (1:1,000 dilution; Santa Cruz Biotechnology catalog no. sc-94), and arginase 1 (1:1,000 dilution; Santa Cruz Biotechnology catalog no. sc-18354).

    Techniques: Activation Assay, Positive Control, Western Blot, Staining, Fluorescence, Confocal Microscopy, Translocation Assay