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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
    https://www.bioz.com/result/monoclonal anti map kinase activated diphosphorylated erk 1 2 antibody/product/Millipore
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    99
    Cell Signaling Technology Inc 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
    Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phosphorylated erk
    Quantification from western blots of phosphorylated to total <t>Akt</t> (A) and phosphorylated to total <t>ERK</t> (C) in 2–4 day old isolated cardiomyocytes from control and sertraline-exposed mice at baseline and after stimulation with 5-HT (B,D). N= 11 saline, 8 sertraline. *p
    Phosphorylated Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology phospho erk
    Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of <t>PAP.</t> PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by <t>ERK</t> in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).
    Phospho Erk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1029 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho erk 1 2
    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 
    Phospho Erk 1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology phosphorylated erk
    Comparison of mRNA expressions and the correlations among of <t>Raf,</t> MEK, and <t>ERK.</t> A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, 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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    99
    Cell Signaling Technology Inc antibody against phospho erk
    Comparison of mRNA expressions and the correlations among of <t>Raf,</t> MEK, and <t>ERK.</t> A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P
    Antibody Against Phospho Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti phosphorylated erk
    Comparison of mRNA expressions and the correlations among of <t>Raf,</t> MEK, and <t>ERK.</t> A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P
    Anti Phosphorylated Erk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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

    SP600125 inhibits JNK phosphorylation. Phosphorylation levels of JNK, ERK, and p38 MAPk following treatment with the inhibitor SP600125 (20 or 35 µM) or the control DMSO and 10 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to stretched DMSO controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels in DMSO treated wells. Both concentrations of SP600125 eliminated significant increases in JNK above unstretched levels. Stretch with a treatment of 35 µM of SP600125 resulted in significantly increased ERK phosphorylation compared to DMSO treated stretched wells. Significance is defined as p

    Journal: PLoS ONE

    Article Title: MAPk Activation Modulates Permeability of Isolated Rat Alveolar Epithelial Cell Monolayers Following Cyclic Stretch

    doi: 10.1371/journal.pone.0010385

    Figure Lengend Snippet: SP600125 inhibits JNK phosphorylation. Phosphorylation levels of JNK, ERK, and p38 MAPk following treatment with the inhibitor SP600125 (20 or 35 µM) or the control DMSO and 10 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to stretched DMSO controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels in DMSO treated wells. Both concentrations of SP600125 eliminated significant increases in JNK above unstretched levels. Stretch with a treatment of 35 µM of SP600125 resulted in significantly increased ERK phosphorylation compared to DMSO treated stretched wells. Significance is defined as p

    Article Snippet: Inhibition of MAP Kinases Following 5 days in culture, wells were washed with Delbecco's Modified Eagle's Medium (DMEM) and serum deprived for 2 hours, then treated for 1 hour with 1.5 mls of DMEM containing either SP600125 (20 or 35 µM) or U0126 (10 or 20 µM) to inhibit JNK or ERK phosphorylation respectively (Cell Signaling, Danvers, MA).

    Techniques:

    Stretch activation of MAPk signaling. Phosphorylation levels of JNK, ERK, and p38 MAPk following 10, 30, and 60 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to unstretched controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels (value of 1) following 10 minutes of stretch (*). Following 30 or 60 minutes of stretch, only ERK remained significantly elevated. JNK phosphorylation was significantly greater following 10 minutes than following 60 minutes (#). At the 10 minute time point, phosphorylation of JNK and ERK was significantly higher than phosphorylation of p38. Significance is defined as p

    Journal: PLoS ONE

    Article Title: MAPk Activation Modulates Permeability of Isolated Rat Alveolar Epithelial Cell Monolayers Following Cyclic Stretch

    doi: 10.1371/journal.pone.0010385

    Figure Lengend Snippet: Stretch activation of MAPk signaling. Phosphorylation levels of JNK, ERK, and p38 MAPk following 10, 30, and 60 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to unstretched controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels (value of 1) following 10 minutes of stretch (*). Following 30 or 60 minutes of stretch, only ERK remained significantly elevated. JNK phosphorylation was significantly greater following 10 minutes than following 60 minutes (#). At the 10 minute time point, phosphorylation of JNK and ERK was significantly higher than phosphorylation of p38. Significance is defined as p

    Article Snippet: Inhibition of MAP Kinases Following 5 days in culture, wells were washed with Delbecco's Modified Eagle's Medium (DMEM) and serum deprived for 2 hours, then treated for 1 hour with 1.5 mls of DMEM containing either SP600125 (20 or 35 µM) or U0126 (10 or 20 µM) to inhibit JNK or ERK phosphorylation respectively (Cell Signaling, Danvers, MA).

    Techniques: Activation Assay

    U0126 inhibits ERK phosphorylation. Phosphorylation levels of JNK, ERK, and p38 MAPk following treatment with the inhibitor U0126 (10 or 20 µM) or the control U0124 (20 µM) and 10 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to stretched U0124 controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels in U0124 treated wells. Following treatment with 10 or 20 µM of U0126 and 10 minutes of stretch, ERK phosphorylation was significantly reduced compared with U0124 treatment and stretch. Only treatment with 20 µM of U0126 eliminated significant increases in ERK above unstretched levels. Significance is defined as p

    Journal: PLoS ONE

    Article Title: MAPk Activation Modulates Permeability of Isolated Rat Alveolar Epithelial Cell Monolayers Following Cyclic Stretch

    doi: 10.1371/journal.pone.0010385

    Figure Lengend Snippet: U0126 inhibits ERK phosphorylation. Phosphorylation levels of JNK, ERK, and p38 MAPk following treatment with the inhibitor U0126 (10 or 20 µM) or the control U0124 (20 µM) and 10 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to stretched U0124 controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels in U0124 treated wells. Following treatment with 10 or 20 µM of U0126 and 10 minutes of stretch, ERK phosphorylation was significantly reduced compared with U0124 treatment and stretch. Only treatment with 20 µM of U0126 eliminated significant increases in ERK above unstretched levels. Significance is defined as p

    Article Snippet: Inhibition of MAP Kinases Following 5 days in culture, wells were washed with Delbecco's Modified Eagle's Medium (DMEM) and serum deprived for 2 hours, then treated for 1 hour with 1.5 mls of DMEM containing either SP600125 (20 or 35 µM) or U0126 (10 or 20 µM) to inhibit JNK or ERK phosphorylation respectively (Cell Signaling, Danvers, MA).

    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

    Quantification from western blots of phosphorylated to total Akt (A) and phosphorylated to total ERK (C) in 2–4 day old isolated cardiomyocytes from control and sertraline-exposed mice at baseline and after stimulation with 5-HT (B,D). N= 11 saline, 8 sertraline. *p

    Journal: Journal of cardiovascular pharmacology

    Article Title: Cardiac Outcomes after Perinatal Sertraline Exposure in Mice

    doi: 10.1097/FJC.0000000000000501

    Figure Lengend Snippet: Quantification from western blots of phosphorylated to total Akt (A) and phosphorylated to total ERK (C) in 2–4 day old isolated cardiomyocytes from control and sertraline-exposed mice at baseline and after stimulation with 5-HT (B,D). N= 11 saline, 8 sertraline. *p

    Article Snippet: Primary antibodies for β-actin (1:10000), total (1:5000) and phosphorylated Akt (1:1000) and total (1:2000) and phosphorylated ERK (1:2000)(Cell Signaling Technology, Danvers, MA) were assessed.

    Techniques: Western Blot, Isolation, Mouse Assay

    Western blot analysis after β-escin treatment. HUVECs were pre-treated with the indicated concentration of β-escin and then stimulated with 30 ng/mL of bFGF for 30 min before collection. Phosphorylated Akt, ERK 1/2 or p38 were detected by specific antibodies. The pictures shown are representative of three independent experiments. Western blots were quantified by densitometry and the ratio of phosphorylated Akt, phosphorylated ERK 1/2 or phosphorylated p38 to their total counterpart was expressed as mean ± SD of three experiments (***  p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: β-Escin Effectively Modulates HUVECs Proliferation and Tube Formation

    doi: 10.3390/molecules23010197

    Figure Lengend Snippet: Western blot analysis after β-escin treatment. HUVECs were pre-treated with the indicated concentration of β-escin and then stimulated with 30 ng/mL of bFGF for 30 min before collection. Phosphorylated Akt, ERK 1/2 or p38 were detected by specific antibodies. The pictures shown are representative of three independent experiments. Western blots were quantified by densitometry and the ratio of phosphorylated Akt, phosphorylated ERK 1/2 or phosphorylated p38 to their total counterpart was expressed as mean ± SD of three experiments (*** p

    Article Snippet: The following primary antibodies were used: anti-phospho-ERK 1/2 (Cell Signaling Technology, Beverly, MA, USA, 1:2000), anti-phospho-Akt (Cell Signaling Technology, 1:2000) and anti-phospho-p38 (Cell Signaling Technology, 1:1000).

    Techniques: Western Blot, Concentration Assay

    Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of PAP. PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by ERK in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Generation of anti-phosphoserine 537 antibody. ( A ) The position of serine 537 is shown in exons of PAP. PAP I and PAP II share the region exons 1–19. ( B ) Amino acid sequences near 537 serine are conserved in vertebrate PAPs. The sequences of frog, cow, human, chicken and mouse PAP were shown aligned. The best alignments for PAP paralogs, PAPβ and PAPγ were also shown. ( C ) Antisera against the phosphospecific-peptide corresponding to 10 amino acids around serine 537 was generated from rat. The GST-488-542 PAP derivative (lanes 1, 2, 5 and 6) and mutant S537A (lanes 3, 4, 7 and 8) were purified from E. coli and phosphorylated by ERK in vitro using 10 mM ATP. Odd-numbered lanes, ERK-treated; even-numbered lanes, no ERK-treated. The products were analyzed by immunoblot with anti-phosphoserine 537 (lanes 1–4) or pre-immune serum for control (lanes 5–8).

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Papanicolaou Stain, Generated, Mutagenesis, Purification, In Vitro

    Phosphorylation of serine 537 by stimulation of ERK in vivo . ( A ) HeLa cells were transfected with GST–CTD (residues 472–739) (lanes 1–6) and its S537 mutant (lanes 7–9). Cells were cultured in serum free media for 12 h and then stimulated with 20% serum (lanes 1–3 and 7–9) or 20% serum containing PD98059 (50 μM) (lanes 4–6) for the indicated time (lanes 1, 4 and 7, 0 min; lanes 2, 5 and 8, 5 min; lanes 3, 6 and 9, 10 min). To examine the phosphorylation status of serine 537, GST pull-downed complexes were visualized by immunoblot with anti-phosphoserine 537 antiserum. Activation of ERK in total lysates was confirmed by immunoblot with anti-p-ERK antibody. The phosphorylation signals were quantified against the amount of GST-CTD treated with λ protein phosphatase, and shown below the the immnoblots. ( B ) HeLa cells were transfected with full-length GST-PAP. PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml at 12 h after transfection and the cells were further incubated (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min). GST pull-downed complexes from the cell lysates were visualized by immunoblot with anti-phosphoserine 537 antiserum. The amounts of GST-CTD present in the lysates were evaluated with anti-GST antibody after the treatment with λ protein phosphatase.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of serine 537 by stimulation of ERK in vivo . ( A ) HeLa cells were transfected with GST–CTD (residues 472–739) (lanes 1–6) and its S537 mutant (lanes 7–9). Cells were cultured in serum free media for 12 h and then stimulated with 20% serum (lanes 1–3 and 7–9) or 20% serum containing PD98059 (50 μM) (lanes 4–6) for the indicated time (lanes 1, 4 and 7, 0 min; lanes 2, 5 and 8, 5 min; lanes 3, 6 and 9, 10 min). To examine the phosphorylation status of serine 537, GST pull-downed complexes were visualized by immunoblot with anti-phosphoserine 537 antiserum. Activation of ERK in total lysates was confirmed by immunoblot with anti-p-ERK antibody. The phosphorylation signals were quantified against the amount of GST-CTD treated with λ protein phosphatase, and shown below the the immnoblots. ( B ) HeLa cells were transfected with full-length GST-PAP. PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml at 12 h after transfection and the cells were further incubated (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min). GST pull-downed complexes from the cell lysates were visualized by immunoblot with anti-phosphoserine 537 antiserum. The amounts of GST-CTD present in the lysates were evaluated with anti-GST antibody after the treatment with λ protein phosphatase.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: In Vivo, Transfection, Mutagenesis, Cell Culture, Activation Assay, Incubation

    Analysis of phosphorylation site. ( A ) MS/MS spectrum of the doubly charged ion of the peptide including S537. CTD of PAP (residues 488–739) was overexpressed in E. coli and purified. The purified CTD was phosphorylated by ERK using 10 mM ATP in vitro . The untreated (control) and phosphorylated CTD forms were excised from gel and compared by Q-TOF MS/MS analysis. The sequence annotations above the spectra correspond to the y-ion fragment series. The sequences (C- to N-terminus) within the spectrum correspond to the signals indicated by the vertical lines. ( B ) The wild-type GST-CTD (lane 1) and mutants S537A (lane 2) and S534A (lane 3) were expressed in E. coli and purified. The purified GST–CTD proteins were phosphorylated by ERK in vitro using [γ- 32 P]ATP. Phosphorylated band were visualized by autoradiography after SDS-PAGE. Input proteins (200 ng) were also visualized by Coomassie staining.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Analysis of phosphorylation site. ( A ) MS/MS spectrum of the doubly charged ion of the peptide including S537. CTD of PAP (residues 488–739) was overexpressed in E. coli and purified. The purified CTD was phosphorylated by ERK using 10 mM ATP in vitro . The untreated (control) and phosphorylated CTD forms were excised from gel and compared by Q-TOF MS/MS analysis. The sequence annotations above the spectra correspond to the y-ion fragment series. The sequences (C- to N-terminus) within the spectrum correspond to the signals indicated by the vertical lines. ( B ) The wild-type GST-CTD (lane 1) and mutants S537A (lane 2) and S534A (lane 3) were expressed in E. coli and purified. The purified GST–CTD proteins were phosphorylated by ERK in vitro using [γ- 32 P]ATP. Phosphorylated band were visualized by autoradiography after SDS-PAGE. Input proteins (200 ng) were also visualized by Coomassie staining.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Mass Spectrometry, Purification, In Vitro, Sequencing, Autoradiography, SDS Page, Staining

    Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of PAP by ERK in vitro . Lysates of HeLa cells transfected with ( A ) full-length GST-PAP or ( B ) GST-PAP truncation derivatives were incubated with glutathione-Sepharose beads. Glutathione beads were phosphorylated by constitutively active kinases in the presence of [γ- 32 P]ATP for 1 h at 37°C. Sample buffer was added to stop the reaction and proteins were separated by SDS-PAGE. Phosphorylated bands were visualized by autoradiography. Expression of PAP derivatives were confirmed by immunoblot on the lysates with GST antibody. ( C ) GST fusion derivatives were expressed in E. coli . Fusion proteins were eluted from glutathione-Sepharose beads and each proteins were phosphorylated by constitutively active ERK in the presence of [γ- 32 P]ATP for 1 h at 37°C. The products were analyzed as in (B) and input proteins were also visualized by Coomassie staining. ( D ) Data of (B) and (C) are schematically presented to show the region of PAP required for the phosphorylation by ERK.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: In Vitro, Transfection, Incubation, SDS Page, Autoradiography, Expressing, Staining

    Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Effect of phosphorylation of serine 537 on nonspecific activity of PAP. ( A ) HeLa cells were transfected with fusions of Flag-PAP wt (lanes 2–4) or S537A (lanes 5–7) and cell lysates were treated with λ-protein phosphatase. Flag-PAPs were precipitated by incubating cell lysates with protein G agarose beads and anti-Flag antibody. Flag-PAP-bound beads were phosphorylated with the indicated amounts of ERK (lanes 2 and 5, 0 ng/μl; lanes 3 and 6, 1 ng/μl; lanes 4 and 7, 2 ng/μl) in vitro and used for the in vitro polyadenylation assay. We determined the activity of Flag-PAP by measuring incorporation of AMP from [α- 32 P] ATP into 120 nt-length RNA. Polyadenylated RNAs were analyzed on a 5% polyacrylamide gel containing 8 M urea (left). The 120 nt-length RNA labeled at the 3′ end with [ 32 P]pCp was electrophoresed as a control (lane 1). The Flag-PAP present in the purified fraction was semiquantitatively determined by immunoblotting with anti-Flag. Phosphorylation of PAP at serine 537 was visualized by immunoblot with anti-phospho-537 serine. Alternatively, polyadenylated RNAs were separated from free [α- 32 P] ATP in PEI membrane by TLC and analyzed by scintillation (right). Relative PAP activities are expressed as the quantities of polyadenylated RNAs relative to that of RNA polyadenylated by Flag-PAP(wt) alone after normalization to imunoblot signals of PAP. The values are calculated from three independent experiments.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Activity Assay, Transfection, Papanicolaou Stain, In Vitro, Labeling, Purification, Thin Layer Chromatography

    Phosphorylation of serine 537 of endogenous PAP. HeLa cells were cultured in media with 10% serum. When cell confluence reaches 70–80%, PMA was added to 100 ng/ml. At 10 min after the PMA treatment, cell extracts were prepared. They were immunoprecipitated with anti-phosphoserine 537 antibody (lane 2) or pre-immune serum (lane 3). The immunoprecipitates were blotted with anti-PAP antibody. Lysates (5% input) alone (lane 1) or anti-phosphoserine 537 antibody alone (lane 4) before the immunoprecipitation was electrophoresed and blotted with anti-PAP antibody for controls. ( B ) Alternatively, cell extracts were prepared at the indicated time (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min) after PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml. The lysates were blotted with anti-phosphoserine 537, anti-PAP, anti-pERK or anti-ERK antibody.

    Journal: Nucleic Acids Research

    Article Title: ERK is a novel regulatory kinase for poly(A) polymerase

    doi: 10.1093/nar/gkm1091

    Figure Lengend Snippet: Phosphorylation of serine 537 of endogenous PAP. HeLa cells were cultured in media with 10% serum. When cell confluence reaches 70–80%, PMA was added to 100 ng/ml. At 10 min after the PMA treatment, cell extracts were prepared. They were immunoprecipitated with anti-phosphoserine 537 antibody (lane 2) or pre-immune serum (lane 3). The immunoprecipitates were blotted with anti-PAP antibody. Lysates (5% input) alone (lane 1) or anti-phosphoserine 537 antibody alone (lane 4) before the immunoprecipitation was electrophoresed and blotted with anti-PAP antibody for controls. ( B ) Alternatively, cell extracts were prepared at the indicated time (lanes 1 and 4, 0 min; lanes 2 and 5, 5 min; lanes 3 and 6, 10 min) after PMA alone (lanes 1–3) or with PD98059 (50 μM) (lanes 4–6) was added to 100 ng/ml. The lysates were blotted with anti-phosphoserine 537, anti-PAP, anti-pERK or anti-ERK antibody.

    Article Snippet: Antibodies specific for PAP (Santa Cruz), GST (Santa Cruz), ERK (Santa Cruz), phospho-ERK (Santa Cruz), phospho-serine (Chemicon) and Flag (Sigma) were purchased.

    Techniques: Cell Culture, Immunoprecipitation

    Fig. 6. MEK* and ΔRaf trigger MAPK activation when phosphatases are inhibited by okadaic acid (OA). ( A ) ERK immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 6. MEK* and ΔRaf trigger MAPK activation when phosphatases are inhibited by okadaic acid (OA). ( A ) ERK immunoblot. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and treated with both puromycin and OA, where they resumed meiosis. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lane 1, control GV oocytes; lanes 2, 3 and 4, oocytes matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( B ) MBP kinase assay. Groups of 10 oocytes were subjected to MBP kinase assay. Oocytes, matured in puromycin- and OA-containing medium and collected 1.5 h after GVBD, were either not injected (lane 2), or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4). ( C ) MEK* triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2), or injected with MEK* (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. ( D ) ΔRaf triggers MAPK activation in mos –/– oocytes that were cultured in OA. Mos –/– oocytes were either not injected (lanes 1 and 2) or injected with ΔRaf (lanes 3 and 4), cultured for 5 h in dbcAMP, released in M2 medium for overnight culture and then cultured for 1.5 h with (+) or without (–) OA. Lane 5: control M II-arrested oocytes. Groups of 25 oocytes were immunoblotted with the anti-ERK serum.

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Activation Assay, Injection, Cell Culture, Kinase Assay

    Fig. 3. Overexpression of ΔRaf does not induce MAPK activation in puromycin-treated oocytes. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and incubated in 10 µg/ml puromycin. Batches of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 5, respectively, control GV oocytes and oocytes matured for 1.5 h post-GVBD in M2 medium; lanes 2, 3 and 4, oocytes matured in puromycin-containing medium and collected 1.5 h post-GVBD, either not injected (lane 2) or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4).

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 3. Overexpression of ΔRaf does not induce MAPK activation in puromycin-treated oocytes. Oocytes were injected with either full-length Raf1 (as an injection control) or ΔRaf, cultured for 12 h in dbcAMP, then washed from dbcAMP and incubated in 10 µg/ml puromycin. Batches of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 5, respectively, control GV oocytes and oocytes matured for 1.5 h post-GVBD in M2 medium; lanes 2, 3 and 4, oocytes matured in puromycin-containing medium and collected 1.5 h post-GVBD, either not injected (lane 2) or injected with full-length Raf1 (lane 3) or ΔRaf (lane 4).

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Over Expression, Activation Assay, Injection, Cell Culture, Incubation

    Fig. 8. The co-injection of MEK* and Xp42 mapk D324N in mos –/– oocytes rescues the M II arrest. ( A ) Mos –/– oocytes were either not injected (control), injected with RNAs encoding Xp42 mapk D324N alone (xMAPK*), co-injected with wild-type MycERK2 and constitutively active MEK* (MAPK + MEK*), or co-injected with RNAs encoding Xp42 mapk D324N and constitutively active MEK* (xMAPK* + MEK*) in dbcAMP-containing medium. After 5 h incubation in dbcAMP to allow overexpression of the exogenous proteins, the oocytes were transferred to M2 medium for overnight culture. The oocytes were then scored for the presence of polar bodies and normal bipolar spindles (see B): 0 or 1 polar body with abnormal spindles (arrested in M I or M II, abnormal spindles); one polar body and a normal spindle (M II); or two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. ( B ) Immunofluorescence staining of microtubules and chromatin in mos –/– oocytes microinjected with MEK* and Xp42 mapk D324N (MEK* + xMAPK*) or Xp42 mapk D324N alone (xMAPK*). Oocytes were microinjected in dbcAMP and kept in the drug for 5 h. The oocytes were transferred to M2 medium for overnight culture and fixed 16 h after GVBD. Microtubules appear in green and chromosomes in red. Top: normal spindles in oocytes injected with MEK* and Xp42 mapk D324N that extruded one polar body (M II). Bottom: abnormal spindles in oocytes injected with Xp42 mapk D324N that did not extrude the first polar body (M I, left) or extruded only one polar body (M II, right). ( C ) M II-arrested mos –/– oocytes co-injected with MEK* and Xp42 mapk D324N show Xp42 mapk D324N phosphorylation whereas the activated ones do not. Oocytes treated as in (A) were collected separately, i.e. M II-arrested (M II, lane 1) and activated (PB2, lane 2). Groups of 15 oocytes were immunoblotted with the anti-ERK serum. These results correspond to two independent experiments. The MycERK2 is overexpressed after co-injection with MEK* into mos –/– oocytes. Fifty mos –/– oocytes either not injected (NI, lane 3) or co-injected with MEK* and MycERK2 (MEK* + MycERK2, lane 4) were scored after second polar body extrusion, then collected and subjected to immunoblotting using the anti-Myc antibody.

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 8. The co-injection of MEK* and Xp42 mapk D324N in mos –/– oocytes rescues the M II arrest. ( A ) Mos –/– oocytes were either not injected (control), injected with RNAs encoding Xp42 mapk D324N alone (xMAPK*), co-injected with wild-type MycERK2 and constitutively active MEK* (MAPK + MEK*), or co-injected with RNAs encoding Xp42 mapk D324N and constitutively active MEK* (xMAPK* + MEK*) in dbcAMP-containing medium. After 5 h incubation in dbcAMP to allow overexpression of the exogenous proteins, the oocytes were transferred to M2 medium for overnight culture. The oocytes were then scored for the presence of polar bodies and normal bipolar spindles (see B): 0 or 1 polar body with abnormal spindles (arrested in M I or M II, abnormal spindles); one polar body and a normal spindle (M II); or two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. ( B ) Immunofluorescence staining of microtubules and chromatin in mos –/– oocytes microinjected with MEK* and Xp42 mapk D324N (MEK* + xMAPK*) or Xp42 mapk D324N alone (xMAPK*). Oocytes were microinjected in dbcAMP and kept in the drug for 5 h. The oocytes were transferred to M2 medium for overnight culture and fixed 16 h after GVBD. Microtubules appear in green and chromosomes in red. Top: normal spindles in oocytes injected with MEK* and Xp42 mapk D324N that extruded one polar body (M II). Bottom: abnormal spindles in oocytes injected with Xp42 mapk D324N that did not extrude the first polar body (M I, left) or extruded only one polar body (M II, right). ( C ) M II-arrested mos –/– oocytes co-injected with MEK* and Xp42 mapk D324N show Xp42 mapk D324N phosphorylation whereas the activated ones do not. Oocytes treated as in (A) were collected separately, i.e. M II-arrested (M II, lane 1) and activated (PB2, lane 2). Groups of 15 oocytes were immunoblotted with the anti-ERK serum. These results correspond to two independent experiments. The MycERK2 is overexpressed after co-injection with MEK* into mos –/– oocytes. Fifty mos –/– oocytes either not injected (NI, lane 3) or co-injected with MEK* and MycERK2 (MEK* + MycERK2, lane 4) were scored after second polar body extrusion, then collected and subjected to immunoblotting using the anti-Myc antibody.

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Injection, Incubation, Over Expression, Immunofluorescence, Staining

    Fig. 4. ( A ) Overexpression of ΔRaf does not induce MAPK activation in mos –/– oocytes while Mos overexpression does. Mos –/– oocytes were injected with mRNAs encoding either full-length Raf1 (as an injection control), ΔRaf or Mos, cultured for 5 h in dbcAMP then removed from dbcAMP and collected at various times after GVBD. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 2, respectively, Raf1- and ΔRaf-injected mos –/– oocytes collected 3 h after GVBD; lanes 3 and 4, control mos +/– oocytes matured for 2 (lane 3) or 12 h (lane 4) post-GVBD; lanes 5 and 6, respectively, non-injected and Mos-injected mos –/– oocytes collected 3 h after GVBD. ( B ) Mos, but not MEK* or ΔRaf, restores the M II arrest in mos –/– oocytes. Mos –/– oocytes were either not injected (Control) or were injected with RNAs encoding full-length Mos, constitutively active MEK* or ΔRaf in dbcAMP-containing medium. The oocytes were kept for 5 h in this medium and released in M2 medium for overnight culture. We then scored the oocytes with no polar body (MI), with only one polar body (M II) or with two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. These results correspond to at least three independent experiments. ( C ) Mos –/– oocytes injected with Mos and arrested in M II (top) and spontaneously activated control non-injected oocytes with two polar bodies (bottom).

    Journal: The EMBO Journal

    Article Title: Mos activates MAP kinase in mouse oocytes through two opposite pathways

    doi: 10.1093/emboj/19.22.6065

    Figure Lengend Snippet: Fig. 4. ( A ) Overexpression of ΔRaf does not induce MAPK activation in mos –/– oocytes while Mos overexpression does. Mos –/– oocytes were injected with mRNAs encoding either full-length Raf1 (as an injection control), ΔRaf or Mos, cultured for 5 h in dbcAMP then removed from dbcAMP and collected at various times after GVBD. Groups of 25 oocytes were immunoblotted with the anti-ERK serum. Lanes 1 and 2, respectively, Raf1- and ΔRaf-injected mos –/– oocytes collected 3 h after GVBD; lanes 3 and 4, control mos +/– oocytes matured for 2 (lane 3) or 12 h (lane 4) post-GVBD; lanes 5 and 6, respectively, non-injected and Mos-injected mos –/– oocytes collected 3 h after GVBD. ( B ) Mos, but not MEK* or ΔRaf, restores the M II arrest in mos –/– oocytes. Mos –/– oocytes were either not injected (Control) or were injected with RNAs encoding full-length Mos, constitutively active MEK* or ΔRaf in dbcAMP-containing medium. The oocytes were kept for 5 h in this medium and released in M2 medium for overnight culture. We then scored the oocytes with no polar body (MI), with only one polar body (M II) or with two polar bodies (spontaneously activated oocytes, PB2). The numbers in parentheses represent the total number of oocytes injected. These results correspond to at least three independent experiments. ( C ) Mos –/– oocytes injected with Mos and arrested in M II (top) and spontaneously activated control non-injected oocytes with two polar bodies (bottom).

    Article Snippet: The phosphorylated form of MAPK was detected using an anti-phospho-ERK (SC 7383, Santa Cruz Biotechnology).

    Techniques: Over Expression, Activation Assay, Injection, Cell Culture

    sHz activates ERK and NF-κB signaling pathways independent of MyD88 ( A ) Thioglycolate elicited peritoneal macrophages from WT C57BL/6 mice were stimulated with 100 μg of sHz and the activation of the ERK1/2, JNK and p38 MAPK pathways was assessed by western blotting for phosphorylation of ERK1/2, JNK and p38 ( upper panels ) compared to total protein controls ( lower panels ). ( B ) Thioglycolate elicited peritoneal macrophages from MyD88−/− and TLR9−/− C57BL/6 mice were stimulated with 100 μg of sHz and the activation of the ERK1/2 pathway was assessed by western blotting for phosphorylation of ERK1/2 ( upper panels ) compared to total ERK protein ( lower panels ). ( C ) RAW 264.7 cells with a stable NF-κB luciferase promoter were stimulated with 100 ug/ml of sHz in the presence or absence of IFN-γ for 12 hrs. For control versus sHz stimulation: **P

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Pure hemozoin is inflammatory in vivo and activates the NALP3 inflammasome via release of uric acid

    doi: 10.4049/jimmunol.0713552

    Figure Lengend Snippet: sHz activates ERK and NF-κB signaling pathways independent of MyD88 ( A ) Thioglycolate elicited peritoneal macrophages from WT C57BL/6 mice were stimulated with 100 μg of sHz and the activation of the ERK1/2, JNK and p38 MAPK pathways was assessed by western blotting for phosphorylation of ERK1/2, JNK and p38 ( upper panels ) compared to total protein controls ( lower panels ). ( B ) Thioglycolate elicited peritoneal macrophages from MyD88−/− and TLR9−/− C57BL/6 mice were stimulated with 100 μg of sHz and the activation of the ERK1/2 pathway was assessed by western blotting for phosphorylation of ERK1/2 ( upper panels ) compared to total ERK protein ( lower panels ). ( C ) RAW 264.7 cells with a stable NF-κB luciferase promoter were stimulated with 100 ug/ml of sHz in the presence or absence of IFN-γ for 12 hrs. For control versus sHz stimulation: **P

    Article Snippet: Equal amounts of protein were electrophoresed on SDS-PAGE gels and Western blotted with anti-phospho-ERK antibody (Santa Cruz Biotechnology) or anti-phospho p38, anti-phospho JNK, or anti-Iκ-B antibodies (Cell Signaling).

    Techniques: Mouse Assay, Activation Assay, Western Blot, Luciferase

    BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition.  a  and  b  Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) protect against OGD induced neuronal death through ERK 1/2 activation and PTEN lipid phosphatase activity inhibition. a and b Western blots analysis of p-AKT ( a ) and p-ERK 1/2 ( b ) levels in cultured primary neurons, bpV(pic) (200 nM) againsts the OGD-induced p-AKT and p-ERK 1/2 down-regulation. Quantification analysis of the levels are on the right (n = 6 independent cultures, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activation Assay, Activity Assay, Inhibition, Western Blot, Cell Culture

    The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: The mechanism of bpV(pic)-mediateded neuroprotect in ischaemia–reperfusion cerebral injury. After ischaemia–reperfusion, the phospho-AKT (Ser 473 ) and phospho-ERK 1/2 (Thr 202 /Tyr 204 ) were down-regulated, inducing the increase of neuronal death and cerebral injury (left). When treated with bpV(pic), we found that bpV(pic) can not only enhance the level of p-AKT and p-ERK 1/2 through inhibiting PTEN lipid phosphatase activity, but also in a PTEN independent pathway to up regulation of ERK 1/2 activity, leading to neuronal survival and animal functional recovery

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activity Assay, Functional Assay

    BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals.  a  Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126.  b  Quantification analysis of the infarct volume [n = 6, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) through PTEN inhibition and ERK 1/2 activation reduces the infarct volume in ischemic stroke animals. a Sample images of TTC staining brain sections show that bpV(pic) decreases the infarct volume in brain 24 h after ischemia onset was prevented by IV and U0126. b Quantification analysis of the infarct volume [n = 6, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Inhibition, Activation Assay, Staining

    BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation.  a  Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) induces the functional recovery in ischemic stroke animals through PTEN inhibition and ERK 1/2 activation. a Animals treated with bpV(pic) have lower scores in mNSS test at day 7 and 14 after ischemia–reperfusion injury compared with I/R + Vehicle group. Animals injected with IV and/or U0126 before injected with bpV(pic) show a higher scores in mNSS test at day 7 and 14 after ischemia–reperfusion than I/R + bpV(pic) group [n = 6 for each group, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Functional Assay, Inhibition, Activation Assay, Injection

    After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased.  a  and  b  Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm.  c  and  d  Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: After ischemic stroke p-AKT and P-ERK 1/2 levels are decreased. a and b Double-immunofluorescence staining of p-AKT or p-ERK 1/2 with NeuN in the peri-infarct area of cortex 24 h or 72 h after I/R compared with the ipsilateral sham, NeuN performes green, P-AKT and p-ERK 1/2 is shown in red and hochest is shown in blue. Scale bar, 20 µm. c and d Western blots showing a decreasing expression in p-AKT ( c ) and p-ERK 1/2 ( d ) at the indicated time points after I/R at rats (left). Right: quantification analysis of normalized p-AKT and p-ERK 1/2 levels (n = 6 per time points, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Double Immunofluorescence Staining, Western Blot, Expressing

    BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury.  a  A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure.  b  and  c  Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) up-regulated the p-AKT and p-ERK 1/2 level in rats and protects against ischemia–reperfusion injury. a A time points diagram shows rat ischemia–reperfusion injury and IV (AKT inhibitor), U0126 (ERK 1/2 inhibitor), bpV(pic) treatment procedure. b and c Western blots showing an increased expression in p-AKT ( b ) and p-ERK 1/2 ( c ) after i.c.v inject bpV(pic) (100 µM, 5 µL) 24 h after ischemia–reperfusion injury comparing with I/R + vehicle group (left). Right: quantification analysis of p-AKT and p-ERK 1/2 levels (n = 6, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Western Blot, Expressing

    BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level.  a  Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Journal: Neurochemical Research

    Article Title: ERK 1/2 Activation Mediates the Neuroprotective Effect of BpV(pic) in Focal Cerebral Ischemia–Reperfusion Injury

    doi: 10.1007/s11064-018-2558-z

    Figure Lengend Snippet: BpV(pic) not only through inhibit PTEN lipid phosphatase activity but also independently of PTEN to up-regulation p-ERK 1/2 level. a Western blots analysis of p-ERK 1/2 levels in SH-SY5Y cells treated with bpV(pic) (10–500 nM) on right. Left: quantification analysis of p-ERK 1/2 levels treated with bpV(pic) shows an increased expression of normalized p-ERK 1/2 compare with vehicle group (n = 6 independent cultures, *P 

    Article Snippet: The brain sections were treated with primary antibody rabbit anti- phospho-AKT (Ser473 ) (1:250), phospho-ERK 1/2 (Thr202 /Tyr204 ) (1:250) from Cell Signaling Technology, mouse anti- NeuN (neuronal-specific nuclear protein) from Chemicon.

    Techniques: Activity Assay, Western Blot, Expressing

    Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P

    Journal: Technology in Cancer Research & Treatment

    Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

    doi: 10.1177/1533034617754024

    Figure Lengend Snippet: Comparison of mRNA expressions and the correlations among of Raf, MEK, and ERK. A, Comparison of mRNA expressions of Raf, MEK, and ERK; *, compared with the normal group, P

    Article Snippet: Citrate buffer (pH 6.0) at high pressure conditions was used to repair antigens (2 minutes) and subsequently cooled off at room temperature with normal goat serum applied overnight (40 minutes), and next, Raf (1:400), MEK (1:400), phosphorylated (p)-MEK (1:400), ERK (1:200), and phosphorylated ERK (p-ERK; 1:200) antibodies (all purchased from SANTA CRUZ Biotechnology, Inc, Santa Cruz, California) were added to incubate paraffin sections at low temperature overnight, respectively.

    Techniques:

    Correlation between protein expressions of Raf, MEK, p-MEK, ERK, and p-ERK and prognosis of patients with BC having ALNM. A, Survival curve of patients with positive and negative protein expression of Raf. B, Survival curve of patients with positive and negative protein expression of MEK. C, Survival curve of patients with positive and negative protein expression of p-MEK. D, Survival curve of patients with positive and negative protein expression of ERK. E, Survival curve of patients with positive and negative protein expression of p-ERK. ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Journal: Technology in Cancer Research & Treatment

    Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

    doi: 10.1177/1533034617754024

    Figure Lengend Snippet: Correlation between protein expressions of Raf, MEK, p-MEK, ERK, and p-ERK and prognosis of patients with BC having ALNM. A, Survival curve of patients with positive and negative protein expression of Raf. B, Survival curve of patients with positive and negative protein expression of MEK. C, Survival curve of patients with positive and negative protein expression of p-MEK. D, Survival curve of patients with positive and negative protein expression of ERK. E, Survival curve of patients with positive and negative protein expression of p-ERK. ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Article Snippet: Citrate buffer (pH 6.0) at high pressure conditions was used to repair antigens (2 minutes) and subsequently cooled off at room temperature with normal goat serum applied overnight (40 minutes), and next, Raf (1:400), MEK (1:400), phosphorylated (p)-MEK (1:400), ERK (1:200), and phosphorylated ERK (p-ERK; 1:200) antibodies (all purchased from SANTA CRUZ Biotechnology, Inc, Santa Cruz, California) were added to incubate paraffin sections at low temperature overnight, respectively.

    Techniques: Expressing

    Comparison of Raf, MEK, p-MEK, ERK, and p-ERK protein expressions among the normal, non-ALNM, and ALNM groups detected by immunohistochemistry (× 200). ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Journal: Technology in Cancer Research & Treatment

    Article Title: Correlation Between Raf/MEK/ERK Signaling Pathway and Clinicopathological Features and Prognosis for Patients With Breast Cancer Having Axillary Lymph Node Metastasis

    doi: 10.1177/1533034617754024

    Figure Lengend Snippet: Comparison of Raf, MEK, p-MEK, ERK, and p-ERK protein expressions among the normal, non-ALNM, and ALNM groups detected by immunohistochemistry (× 200). ALNM indicates axillary lymph node metastasis; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; p-MEK, phosphorylated MEK; Raf, rapidly accelerated fibrosarcoma.

    Article Snippet: Citrate buffer (pH 6.0) at high pressure conditions was used to repair antigens (2 minutes) and subsequently cooled off at room temperature with normal goat serum applied overnight (40 minutes), and next, Raf (1:400), MEK (1:400), phosphorylated (p)-MEK (1:400), ERK (1:200), and phosphorylated ERK (p-ERK; 1:200) antibodies (all purchased from SANTA CRUZ Biotechnology, Inc, Santa Cruz, California) were added to incubate paraffin sections at low temperature overnight, respectively.

    Techniques: Immunohistochemistry

    Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (** P

    Journal: Breast Cancer Research : BCR

    Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

    doi: 10.1186/s13058-014-0479-6

    Figure Lengend Snippet: Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (** P

    Article Snippet: ECM1, HER3, phosphorylated ERK (p-ERK), ERK, actin, MUC1 and galectin-3 antibodies were obtained from Santa Cruz Biotechnology. c-Raf, p-c-Raf, MEK, p-MEK, Akt, p-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Recombinant, Incubation, Western Blot, shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Extracellular matrix protein 1 augments epidermal growth factor signaling. (A) At 24 hours after seeding, BT-474 trastuzumab-resistant (TR) and BT-474 extracellular matrix protein 1 (ECM1)-expressing cells were treated with anti-ECM1 antibodies (ab; 5 μg/ml). Ten minutes later, cell lysates were analyzed on Western blots. (B) After serum starvation for 24 hours, cells were treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) and epidermal growth factor (EGF; 10 ng/ml). Cell lysates were prepared at the indicated time points and analyzed on Western blots. (C) Total cell lysates were incubated with epidermal growth factor receptor (EGFR) antibodies overnight, and immunoprecipitates (IP) were analyzed on Western blots. IgG, Immunoglobulin G; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1; Vec, Vector. (D) Cells were incubated with 0.5 mg/ml EZ-Link NHS-SS-Biotin for 30 minutes at 4°C. The biotinylated proteins were precipitated by streptavidin, and the precipitates were analyzed on Western blots (IB) using ECM1 antibody (left). Cell surface labeling of ECM1 was conducted by immunostaining without permeabilization (right). Extracellular signal-regulated kinase (ERK) was used as an endogenous negative control protein. DIC, Differential Interference Contrast; GSH, Glutathione.

    Journal: Breast Cancer Research : BCR

    Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

    doi: 10.1186/s13058-014-0479-6

    Figure Lengend Snippet: Extracellular matrix protein 1 augments epidermal growth factor signaling. (A) At 24 hours after seeding, BT-474 trastuzumab-resistant (TR) and BT-474 extracellular matrix protein 1 (ECM1)-expressing cells were treated with anti-ECM1 antibodies (ab; 5 μg/ml). Ten minutes later, cell lysates were analyzed on Western blots. (B) After serum starvation for 24 hours, cells were treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) and epidermal growth factor (EGF; 10 ng/ml). Cell lysates were prepared at the indicated time points and analyzed on Western blots. (C) Total cell lysates were incubated with epidermal growth factor receptor (EGFR) antibodies overnight, and immunoprecipitates (IP) were analyzed on Western blots. IgG, Immunoglobulin G; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1; Vec, Vector. (D) Cells were incubated with 0.5 mg/ml EZ-Link NHS-SS-Biotin for 30 minutes at 4°C. The biotinylated proteins were precipitated by streptavidin, and the precipitates were analyzed on Western blots (IB) using ECM1 antibody (left). Cell surface labeling of ECM1 was conducted by immunostaining without permeabilization (right). Extracellular signal-regulated kinase (ERK) was used as an endogenous negative control protein. DIC, Differential Interference Contrast; GSH, Glutathione.

    Article Snippet: ECM1, HER3, phosphorylated ERK (p-ERK), ERK, actin, MUC1 and galectin-3 antibodies were obtained from Santa Cruz Biotechnology. c-Raf, p-c-Raf, MEK, p-MEK, Akt, p-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Recombinant, Incubation, shRNA, Plasmid Preparation, Labeling, Immunostaining, Negative Control