rabbit anti erk1 2  (Millipore)


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    Structured Review

    Millipore rabbit anti erk1 2
    AAs activate <t>ERK1/2</t> and mTORC1. MIN6 cells (A, C, and D) were placed in KRBH without AAs (see Materials and Methods ) and 4.5 mM glucose for 2–3 hours prior to stimulation with AAs for the times indicated on each panel. Human islets (B) were treated similarly except that 2 mM glucose was used. After treatment, cell lysates were resolved on gels and transferred to nitrocellulose for immunoblotting of pERK1/2, pS6K, pS6 (240/244), or p4EBP1 as indicated. ERK1/2, S6K, and S6 were loading controls used to normalize the p protein signals to total protein amount (eg, pS6K/S6K). The value for the untreated control signal is set to 1. A, Stimulation with AAs for the indicated times and is representative of 10 experiments. Quantification of 4 is shown in Supplemental Figure 1A. B, Stimulation of islets with AAs for 2 minutes. One of two experiments with islets from different donors is shown. C, Stimulation with individual AAs at 0.25, 0.5, or 5 mM for 2 minutes. One of six experiments is shown. D, Stimulation with individual AAs at 1 mM for 30 minutes. Immunoblots show one of three experiments is shown. Bar graphs are means of quantification ± SEM (n = 3). *, P
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    Images

    1) Product Images from "Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells"

    Article Title: Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells

    Journal: Molecular Endocrinology

    doi: 10.1210/ME.2014-1181

    AAs activate ERK1/2 and mTORC1. MIN6 cells (A, C, and D) were placed in KRBH without AAs (see Materials and Methods ) and 4.5 mM glucose for 2–3 hours prior to stimulation with AAs for the times indicated on each panel. Human islets (B) were treated similarly except that 2 mM glucose was used. After treatment, cell lysates were resolved on gels and transferred to nitrocellulose for immunoblotting of pERK1/2, pS6K, pS6 (240/244), or p4EBP1 as indicated. ERK1/2, S6K, and S6 were loading controls used to normalize the p protein signals to total protein amount (eg, pS6K/S6K). The value for the untreated control signal is set to 1. A, Stimulation with AAs for the indicated times and is representative of 10 experiments. Quantification of 4 is shown in Supplemental Figure 1A. B, Stimulation of islets with AAs for 2 minutes. One of two experiments with islets from different donors is shown. C, Stimulation with individual AAs at 0.25, 0.5, or 5 mM for 2 minutes. One of six experiments is shown. D, Stimulation with individual AAs at 1 mM for 30 minutes. Immunoblots show one of three experiments is shown. Bar graphs are means of quantification ± SEM (n = 3). *, P
    Figure Legend Snippet: AAs activate ERK1/2 and mTORC1. MIN6 cells (A, C, and D) were placed in KRBH without AAs (see Materials and Methods ) and 4.5 mM glucose for 2–3 hours prior to stimulation with AAs for the times indicated on each panel. Human islets (B) were treated similarly except that 2 mM glucose was used. After treatment, cell lysates were resolved on gels and transferred to nitrocellulose for immunoblotting of pERK1/2, pS6K, pS6 (240/244), or p4EBP1 as indicated. ERK1/2, S6K, and S6 were loading controls used to normalize the p protein signals to total protein amount (eg, pS6K/S6K). The value for the untreated control signal is set to 1. A, Stimulation with AAs for the indicated times and is representative of 10 experiments. Quantification of 4 is shown in Supplemental Figure 1A. B, Stimulation of islets with AAs for 2 minutes. One of two experiments with islets from different donors is shown. C, Stimulation with individual AAs at 0.25, 0.5, or 5 mM for 2 minutes. One of six experiments is shown. D, Stimulation with individual AAs at 1 mM for 30 minutes. Immunoblots show one of three experiments is shown. Bar graphs are means of quantification ± SEM (n = 3). *, P

    Techniques Used: Atomic Absorption Spectroscopy, Western Blot

    2) Product Images from "Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease"

    Article Title: Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease

    Journal: Brain Sciences

    doi: 10.3390/brainsci10050315

    Effects of LGF on ERK1/2 and CREB phosphorylation in cultured mesencephalic glia. ( A ) shows the effect of LGF on phospho-ERK1/ERK1 and phospho-ERK2/ERK2 ratios in mesencephalic glia maintained in a DM (white bar) ( A ) and in a defined medium with LGF (DM and LGF) for 6 (dots bar) or 24 h (black bar). Note how LGF increased the phospho-ERK1/ERK1 ratio at both experimental times. ( B ) shows the effect of LGF treatment in phospho-CREB protein expression in DM (white bar) and in a defined medium with LGF (DM and LGF) for 6 (dots bar) or 24 h (vertical black lines bar). Note how LGF treatment for 24 h significantly increased phospho-CREB levels. ( C ) shows representative bands of phospho-ERK1/2, ERK1/2, phospho-CREB, and β-actin. Note how ERK1/2 protein levels are unchanged under all experimental conditions. ( D – F ) show how some phospho-CREB-positive cells—( D – F ) in green—were also immunopositive for phospho-ERK1/2 ( D ) (red) and isolectin IB4 ( E ) (blue), as well as how a few isolectin IB4-positive cells were phospho-ERK1/2- and phospho-CREB-positive too ( F ). Results in ( A , B ) represent the mean ± SEM of 5–9 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. DM, + p ≤ 0.05, +++ p ≤ 0.001 vs. 6 h DM and LGF (ANOVA and Newman–Keuls). In ( C ), lane 1 and 4: CM; lane 2 and 5: DM; lane 3: DM and LGF (6 h); and lane 6: DM and LGF (24 h). Scale bar 100 µm.
    Figure Legend Snippet: Effects of LGF on ERK1/2 and CREB phosphorylation in cultured mesencephalic glia. ( A ) shows the effect of LGF on phospho-ERK1/ERK1 and phospho-ERK2/ERK2 ratios in mesencephalic glia maintained in a DM (white bar) ( A ) and in a defined medium with LGF (DM and LGF) for 6 (dots bar) or 24 h (black bar). Note how LGF increased the phospho-ERK1/ERK1 ratio at both experimental times. ( B ) shows the effect of LGF treatment in phospho-CREB protein expression in DM (white bar) and in a defined medium with LGF (DM and LGF) for 6 (dots bar) or 24 h (vertical black lines bar). Note how LGF treatment for 24 h significantly increased phospho-CREB levels. ( C ) shows representative bands of phospho-ERK1/2, ERK1/2, phospho-CREB, and β-actin. Note how ERK1/2 protein levels are unchanged under all experimental conditions. ( D – F ) show how some phospho-CREB-positive cells—( D – F ) in green—were also immunopositive for phospho-ERK1/2 ( D ) (red) and isolectin IB4 ( E ) (blue), as well as how a few isolectin IB4-positive cells were phospho-ERK1/2- and phospho-CREB-positive too ( F ). Results in ( A , B ) represent the mean ± SEM of 5–9 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs. DM, + p ≤ 0.05, +++ p ≤ 0.001 vs. 6 h DM and LGF (ANOVA and Newman–Keuls). In ( C ), lane 1 and 4: CM; lane 2 and 5: DM; lane 3: DM and LGF (6 h); and lane 6: DM and LGF (24 h). Scale bar 100 µm.

    Techniques Used: Cell Culture, Expressing

    3) Product Images from "Downstream-of-FGFR Is a Fibroblast Growth Factor-Specific Scaffolding Protein and Recruits Corkscrew upon Receptor Activation"

    Article Title: Downstream-of-FGFR Is a Fibroblast Growth Factor-Specific Scaffolding Protein and Recruits Corkscrew upon Receptor Activation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.9.3769-3781.2004

    (A) Activation of the MAPK pathway is important but not sufficient to direct cell migration. Activation of the MAPK pathway wasvisualized with anti-dpERK antibodies (subpanels a to d), the lumen of the tracheal system with the 2A12 antibody in red and terminal cells with the anti-DSRF antibody in green (subpanels e and f). Confocal projections are shown for embryos expressing the following transgenes under the control of the pannier Gal4 driver: UAS dof (subpanel a), UAS dof and UAS btl act (subpanel b), UAS dof600 ΔAR and UAS btl act (subpanel c) and UAS dof600Y515F and UAS btl act (subpanel d). (Subpanels e and f) Embryos expressing UAS dof600 ΔAR or UAS dof600Y515F in a dof −/− background under the control of the btl Gal4 driver are shown. (B) Summary table of the capacity of each transgene to activate dpERK in combination with an activated form of btl . The capacity of each dof transgene to rescue cell migration in dof −/− embryos is also shown in the second column.
    Figure Legend Snippet: (A) Activation of the MAPK pathway is important but not sufficient to direct cell migration. Activation of the MAPK pathway wasvisualized with anti-dpERK antibodies (subpanels a to d), the lumen of the tracheal system with the 2A12 antibody in red and terminal cells with the anti-DSRF antibody in green (subpanels e and f). Confocal projections are shown for embryos expressing the following transgenes under the control of the pannier Gal4 driver: UAS dof (subpanel a), UAS dof and UAS btl act (subpanel b), UAS dof600 ΔAR and UAS btl act (subpanel c) and UAS dof600Y515F and UAS btl act (subpanel d). (Subpanels e and f) Embryos expressing UAS dof600 ΔAR or UAS dof600Y515F in a dof −/− background under the control of the btl Gal4 driver are shown. (B) Summary table of the capacity of each transgene to activate dpERK in combination with an activated form of btl . The capacity of each dof transgene to rescue cell migration in dof −/− embryos is also shown in the second column.

    Techniques Used: Activation Assay, Migration, Expressing, Activated Clotting Time Assay

    4) Product Images from "A Mesh-Duox pathway regulates homeostasis in the insect gut"

    Article Title: A Mesh-Duox pathway regulates homeostasis in the insect gut

    Journal: Nature microbiology

    doi: 10.1038/nmicrobiol.2017.20

    The role of Mesh-Arrestin-ERK/JNK-MAPK signaling cascade in DmDuox regulation in Drosophila (a–b) Regulation of the DmDuox gene in the guts of DmERK (a) and DmJNK (b) RNAi flies. (c) Regulation of ROS activity in the guts of DmERK and DmJNK RNAi flies. (d) Enhancement of the gut microbiome in the guts of DmERK and DmJNK RNAi flies. (e–f) Silencing DmArrestins impaired expression of the DmDuox gene (e) and ROS activity (f) in the Drosophila guts. (g) Increasing the burden of gut microbiome in the guts of DmArrestins RNAi flies. (h–i) Assessing the role of the “Arrestin-ERK/JNK” cascade in AaMesh-mediated Duox expression in Drosophila . Both DmArrestin-1 and DmArrestin-2 were silenced by dsRNA transfection in the pAc-DmMesh-trnasfected Drosophila S2 cells. (h) The phosphorylation of DmERK (p-ERK) and DmJNK (p-JNK) was detected by western blotting. (i) The abundance of the DmDuox gene was determined by SYBR Green qPCR and normalized by Drosophila actin ( CG12051 ). (a–g) GFP RNAi flies served as a negative control. (a–b, e) The gene expression was determined by SYBR Green qPCR and normalized against D. melanogaster actin ( CG12051 ). The qPCR primers are described in Supplementary Table 6 . One dot represents one fly gut. The horizontal line represents the mean value of the results. (c, f) The ROS activity was detected by a H 2 O 2 assay. The data are presented as the mean ± S.E.M. (d, g) The burden of gut microbes was determined by a CFU assay. (a–g, i) The data were analyzed using the non-parametric Mann-Whitney test. (a–i) The results were reproduced by at least 3 independent experiments.
    Figure Legend Snippet: The role of Mesh-Arrestin-ERK/JNK-MAPK signaling cascade in DmDuox regulation in Drosophila (a–b) Regulation of the DmDuox gene in the guts of DmERK (a) and DmJNK (b) RNAi flies. (c) Regulation of ROS activity in the guts of DmERK and DmJNK RNAi flies. (d) Enhancement of the gut microbiome in the guts of DmERK and DmJNK RNAi flies. (e–f) Silencing DmArrestins impaired expression of the DmDuox gene (e) and ROS activity (f) in the Drosophila guts. (g) Increasing the burden of gut microbiome in the guts of DmArrestins RNAi flies. (h–i) Assessing the role of the “Arrestin-ERK/JNK” cascade in AaMesh-mediated Duox expression in Drosophila . Both DmArrestin-1 and DmArrestin-2 were silenced by dsRNA transfection in the pAc-DmMesh-trnasfected Drosophila S2 cells. (h) The phosphorylation of DmERK (p-ERK) and DmJNK (p-JNK) was detected by western blotting. (i) The abundance of the DmDuox gene was determined by SYBR Green qPCR and normalized by Drosophila actin ( CG12051 ). (a–g) GFP RNAi flies served as a negative control. (a–b, e) The gene expression was determined by SYBR Green qPCR and normalized against D. melanogaster actin ( CG12051 ). The qPCR primers are described in Supplementary Table 6 . One dot represents one fly gut. The horizontal line represents the mean value of the results. (c, f) The ROS activity was detected by a H 2 O 2 assay. The data are presented as the mean ± S.E.M. (d, g) The burden of gut microbes was determined by a CFU assay. (a–g, i) The data were analyzed using the non-parametric Mann-Whitney test. (a–i) The results were reproduced by at least 3 independent experiments.

    Techniques Used: Activity Assay, Expressing, Transfection, Western Blot, SYBR Green Assay, Real-time Polymerase Chain Reaction, Negative Control, Colony-forming Unit Assay, MANN-WHITNEY

    5) Product Images from "?-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development"

    Article Title: ?-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2010.202

    Loss of krz results in increased levels of dpERK. ( A , B ) Expression of doubly phosphorylated activated ERK (dpERK) in ( A ) stage-4 control and ( B ) krz 1 maternal mutant embryos. Embryos were stained with an antibody specific for the activated form of ERK. ( C ) Average intensity of dpERK expression plotted around the sagittal circumference of the embryos (D, dorsal; A, anterior; V, ventral; P, posterior). ( D , F ) Quantification of the average dpERK intensity ( D ) and average width of dpERK expression ( F ) in 28 Hist-GFP control and 28 krz 1 maternal mutant embryos (au, arbitrary signal intensity units). ( E ) Western blot of protein expression in staged krz 1 maternal mutant embryos and FRT controls. HSP70 antibody was used as loading control.
    Figure Legend Snippet: Loss of krz results in increased levels of dpERK. ( A , B ) Expression of doubly phosphorylated activated ERK (dpERK) in ( A ) stage-4 control and ( B ) krz 1 maternal mutant embryos. Embryos were stained with an antibody specific for the activated form of ERK. ( C ) Average intensity of dpERK expression plotted around the sagittal circumference of the embryos (D, dorsal; A, anterior; V, ventral; P, posterior). ( D , F ) Quantification of the average dpERK intensity ( D ) and average width of dpERK expression ( F ) in 28 Hist-GFP control and 28 krz 1 maternal mutant embryos (au, arbitrary signal intensity units). ( E ) Western blot of protein expression in staged krz 1 maternal mutant embryos and FRT controls. HSP70 antibody was used as loading control.

    Techniques Used: Expressing, Mutagenesis, Staining, Western Blot

    Krz inhibits ERK phosphorylation by MEK. ( A ) S2 cells were transfected with Drosophila ERK–Flag alone or in combination with HA–Krz-R209E, and immunoprecipitated with either anti-Flag or anti-HA beads. 320 ng of purified human MEK2 was added to samples as indicated, and extent of phosphorylation of Drosophila ERK–Flag was then assayed with anti-dpERK antibody by western blot analysis. ( B ) S2 cells were transfected with Drosophila ERK–Flag alone or in the combinations indicated with GFP–Krz, Drosophila MEK–V5 and/or Drosophila HA–Raf. Cells were treated with insulin at a final concentration of 20 μM as indicated. Samples were immunoprecipitated with anti-Flag beads, and extent of phosphorylation of Drosophila ERK–Flag was then assayed with anti-dpERK antibody by western blot analysis. IP, immunoprecipitated samples; IB, immunoblots. ( C ) A model of Krz regulation of RTK signalling. Krz limits the activity of receptor tyrosine kinases (RTKs) by preferentially binding and sequestering an inactive form of ERK. This sequestration prevents ERK from being phosphorylated by the upstream kinase, MEK, and therefore reduces an overall output of signalling downstream of RTKs.
    Figure Legend Snippet: Krz inhibits ERK phosphorylation by MEK. ( A ) S2 cells were transfected with Drosophila ERK–Flag alone or in combination with HA–Krz-R209E, and immunoprecipitated with either anti-Flag or anti-HA beads. 320 ng of purified human MEK2 was added to samples as indicated, and extent of phosphorylation of Drosophila ERK–Flag was then assayed with anti-dpERK antibody by western blot analysis. ( B ) S2 cells were transfected with Drosophila ERK–Flag alone or in the combinations indicated with GFP–Krz, Drosophila MEK–V5 and/or Drosophila HA–Raf. Cells were treated with insulin at a final concentration of 20 μM as indicated. Samples were immunoprecipitated with anti-Flag beads, and extent of phosphorylation of Drosophila ERK–Flag was then assayed with anti-dpERK antibody by western blot analysis. IP, immunoprecipitated samples; IB, immunoblots. ( C ) A model of Krz regulation of RTK signalling. Krz limits the activity of receptor tyrosine kinases (RTKs) by preferentially binding and sequestering an inactive form of ERK. This sequestration prevents ERK from being phosphorylated by the upstream kinase, MEK, and therefore reduces an overall output of signalling downstream of RTKs.

    Techniques Used: Transfection, Immunoprecipitation, Purification, Western Blot, Concentration Assay, Activity Assay, Binding Assay

    Krz preferentially interacts with inactive ERK. ( A ) S2 cells were transfected with HA–Krz, HA–Krz-K336A, HA–Krz-R209E or HA–Krz-R209E-K336A together with Drosophila ERK–Flag. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. ( B ) S2 cells were transfected with Drosophila ERK–Flag alone, or together with HA–Krz-R209E or HA–Krz. Cells were treated with insulin at a final concentration of 20 μM. Samples were immunoprecipitated with anti-Flag or anti-HA beads. Immunoprecipitates were analysed by western blot with anti-HA antibody, anti-total ERK antibody and anti-dpERK antibody. ( C ) S2 cells were transfected with HA–Krz or HA–Krz-R209E together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. ( D ) S2 cells were transfected with HA–Krz and either Drosophila wild-type ERK–Flag or ERK-D334N–Flag. Lysates were immunoprecipitated with anti-HA beads and immunoblotted. ( E ) S2 cells were transfected with HA–Krz together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. IP, immunoprecipitated samples; IB, immunoblots.
    Figure Legend Snippet: Krz preferentially interacts with inactive ERK. ( A ) S2 cells were transfected with HA–Krz, HA–Krz-K336A, HA–Krz-R209E or HA–Krz-R209E-K336A together with Drosophila ERK–Flag. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. ( B ) S2 cells were transfected with Drosophila ERK–Flag alone, or together with HA–Krz-R209E or HA–Krz. Cells were treated with insulin at a final concentration of 20 μM. Samples were immunoprecipitated with anti-Flag or anti-HA beads. Immunoprecipitates were analysed by western blot with anti-HA antibody, anti-total ERK antibody and anti-dpERK antibody. ( C ) S2 cells were transfected with HA–Krz or HA–Krz-R209E together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. ( D ) S2 cells were transfected with HA–Krz and either Drosophila wild-type ERK–Flag or ERK-D334N–Flag. Lysates were immunoprecipitated with anti-HA beads and immunoblotted. ( E ) S2 cells were transfected with HA–Krz together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. IP, immunoprecipitated samples; IB, immunoblots.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Concentration Assay

    6) Product Images from "Unc-51 controls active zone density and protein composition by downregulating ERK signaling"

    Article Title: Unc-51 controls active zone density and protein composition by downregulating ERK signaling

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.3848-08.2009

    Unc-51 inhibits ERK activation Western blot probed using anti-diphospho (activated) ERK, anti-rolled and anti-syntaxin antibody. Activated ERK is increased in unc-51 mutants compared to wild type (n=3, p
    Figure Legend Snippet: Unc-51 inhibits ERK activation Western blot probed using anti-diphospho (activated) ERK, anti-rolled and anti-syntaxin antibody. Activated ERK is increased in unc-51 mutants compared to wild type (n=3, p

    Techniques Used: Activation Assay, Western Blot

    7) Product Images from "Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans"

    Article Title: Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg472

    Fig. 4. Activated FGFR signals protein degradation via SEM-5 and the Raf–MAPK pathway. Animals were grown to adulthood at 16°C and then for an additional 48 h at 25°C. ( A ) Histochemical staining for β-galactosidase activity. Genotypes (all mutations are reduction-of-function alleles) are indicated on the left. ( B ) Immunoblot (monoclonal anti-β-galactosidase) of 146 kDa β-galactosidase fusion protein in 30-worm lysates. Each row, corresponding to the animals with genotypes in (A), shows the kinetics of loss of the 146 kDa band. ( C ) Quantitation of 146 kDa fusion protein by integrating the bands at t = 48 h in (B) and of β-galactosidase activity (means ± SD of triplicate samples) by fluorimetric assay of 10-worm lysates. Data are expressed as a percentage of the value for each genotype prior to temperature upshift. Rows correspond to the genotypes in (A). ( D ) Activation of MPK-1 in clr-1 mutants 4 h after shift to 25°C. Each lane contains lysate of 40 worms. Upper set blotted with monoclonal anti-pTpY-ERK; lower set blotted with polyclonal anti-ERK.
    Figure Legend Snippet: Fig. 4. Activated FGFR signals protein degradation via SEM-5 and the Raf–MAPK pathway. Animals were grown to adulthood at 16°C and then for an additional 48 h at 25°C. ( A ) Histochemical staining for β-galactosidase activity. Genotypes (all mutations are reduction-of-function alleles) are indicated on the left. ( B ) Immunoblot (monoclonal anti-β-galactosidase) of 146 kDa β-galactosidase fusion protein in 30-worm lysates. Each row, corresponding to the animals with genotypes in (A), shows the kinetics of loss of the 146 kDa band. ( C ) Quantitation of 146 kDa fusion protein by integrating the bands at t = 48 h in (B) and of β-galactosidase activity (means ± SD of triplicate samples) by fluorimetric assay of 10-worm lysates. Data are expressed as a percentage of the value for each genotype prior to temperature upshift. Rows correspond to the genotypes in (A). ( D ) Activation of MPK-1 in clr-1 mutants 4 h after shift to 25°C. Each lane contains lysate of 40 worms. Upper set blotted with monoclonal anti-pTpY-ERK; lower set blotted with polyclonal anti-ERK.

    Techniques Used: Staining, Activity Assay, Quantitation Assay, Fluorimetry Assay, Activation Assay

    8) Product Images from "Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6"

    Article Title: Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

    Journal: Endocrinology

    doi: 10.1210/en.2013-1822

    Activation of MAPK in endometrial cells treated with lipopeptide PAMPs. Endometrial epithelial cells (A) and stromal cells (B) were collected 0, 5, 10, 15, 20, or 25 minutes after treatment with 100 ng/mL PAM or 100 ng/mL FSL-1. The protein from the cells was analyzed by SDS-PAGE and immunoblotted with antibodies against total and phosphorylated forms of p38 (t-p38 and p-p38) and ERK1/2 (tERK1/2 and pERK1/2; □, tERK2; ■, pERK2), and α-tubulin as visual confirmation of the precision of protein loading and transfer. The image for each cell type is representative of 3 independent experiments for PAM (left panel) or FSL-1 (right panel), and the histograms represent the mean ± SEM of densitometric analysis of the ratio of phosphorylated p-p38 to t-p38, pERK1 to tERK1 or pERK2 to tERK2, expressed as fold activation compared with time 0. Values differ from time 0 when data were analyzed by ANOVA, using the Dunnett pairwise multiple comparison t test: *, P
    Figure Legend Snippet: Activation of MAPK in endometrial cells treated with lipopeptide PAMPs. Endometrial epithelial cells (A) and stromal cells (B) were collected 0, 5, 10, 15, 20, or 25 minutes after treatment with 100 ng/mL PAM or 100 ng/mL FSL-1. The protein from the cells was analyzed by SDS-PAGE and immunoblotted with antibodies against total and phosphorylated forms of p38 (t-p38 and p-p38) and ERK1/2 (tERK1/2 and pERK1/2; □, tERK2; ■, pERK2), and α-tubulin as visual confirmation of the precision of protein loading and transfer. The image for each cell type is representative of 3 independent experiments for PAM (left panel) or FSL-1 (right panel), and the histograms represent the mean ± SEM of densitometric analysis of the ratio of phosphorylated p-p38 to t-p38, pERK1 to tERK1 or pERK2 to tERK2, expressed as fold activation compared with time 0. Values differ from time 0 when data were analyzed by ANOVA, using the Dunnett pairwise multiple comparison t test: *, P

    Techniques Used: Activation Assay, SDS Page

    Attenuation of endometrial cell responses to lipopeptides by inhibition of MAPK. Endometrial epithelial (A and B) or stromal cells (C and D) were treated for 30 minutes in medium containing vehicle (V), ERK1/2 inhibitor (ERKi) (ERK activation inhibitor peptide I, 10 μM) or p38 inhibitor (p38i) (InSolution SB 203580, 10 μM) and then were cultured in the same treatment for 6 hours in control medium or medium containing 100 ng/mL PAM (A and C) or 100 ng/mL FSL-1 (B and D). Supernatants were harvested to measure the accumulation of IL-6 by ELISA, and results are expressed as a percentage of treatment with PAM (A and C) or FSL-1 (B and D). Data are presented as mean + SEM percentages and represent 3 independent experiments. Values differ from those for PAMP when data were analyzed by ANOVA using the Dunnett pairwise multiple comparison t test: *, P
    Figure Legend Snippet: Attenuation of endometrial cell responses to lipopeptides by inhibition of MAPK. Endometrial epithelial (A and B) or stromal cells (C and D) were treated for 30 minutes in medium containing vehicle (V), ERK1/2 inhibitor (ERKi) (ERK activation inhibitor peptide I, 10 μM) or p38 inhibitor (p38i) (InSolution SB 203580, 10 μM) and then were cultured in the same treatment for 6 hours in control medium or medium containing 100 ng/mL PAM (A and C) or 100 ng/mL FSL-1 (B and D). Supernatants were harvested to measure the accumulation of IL-6 by ELISA, and results are expressed as a percentage of treatment with PAM (A and C) or FSL-1 (B and D). Data are presented as mean + SEM percentages and represent 3 independent experiments. Values differ from those for PAMP when data were analyzed by ANOVA using the Dunnett pairwise multiple comparison t test: *, P

    Techniques Used: Inhibition, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    9) Product Images from "DER signaling restricts the boundaries of the wing field during Drosophila development"

    Article Title: DER signaling restricts the boundaries of the wing field during Drosophila development

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

    doi:

    Loss of DER signaling generates cell fate transformations in the wing disk. ( A ) DNRaf (UAS-DNRaf 3.1 expressed ectopically under the control of en-Gal4 throughout the posterior wing compartment (raised at 25°C). ( Inset ) The posterior to anterior transformation of the wing margin. Similar results were obtained after ectopic expression of UAS-DNRas (a dominant-negative form of Ras1). ( B ), DER tsla / DER co flies raised at 17°C, transferred to 25°C for 24 h at second instar larval stages, and then returned to 17°C (see Materials and Methods ). Wing mirror-image duplication. ( C and D ) Overexpression of DNRaf 3.1 in clones generated by the flip-out technique and induced at 36 ± 12 h after egg laying (see Materials and Methods ). ( C ) Wing mirror-image duplication. ( D ) Notum to wing transformation. ( E ) En (UAS-En) expressed ectopically under the control of en-Gal4 throughout the posterior wing compartment interferes with en . ( F . ( G ) cnk mutant showing a wing-mirror-image duplication (arrowheads). ( H ) Distribution of Wg expression and MAPK activity in early third instar wing discs. MAPK activity, detected with an anti-dpERK antibody (green), is excluded from the wing pouch (arrowhead) and complementary to Wg expression (red).
    Figure Legend Snippet: Loss of DER signaling generates cell fate transformations in the wing disk. ( A ) DNRaf (UAS-DNRaf 3.1 expressed ectopically under the control of en-Gal4 throughout the posterior wing compartment (raised at 25°C). ( Inset ) The posterior to anterior transformation of the wing margin. Similar results were obtained after ectopic expression of UAS-DNRas (a dominant-negative form of Ras1). ( B ), DER tsla / DER co flies raised at 17°C, transferred to 25°C for 24 h at second instar larval stages, and then returned to 17°C (see Materials and Methods ). Wing mirror-image duplication. ( C and D ) Overexpression of DNRaf 3.1 in clones generated by the flip-out technique and induced at 36 ± 12 h after egg laying (see Materials and Methods ). ( C ) Wing mirror-image duplication. ( D ) Notum to wing transformation. ( E ) En (UAS-En) expressed ectopically under the control of en-Gal4 throughout the posterior wing compartment interferes with en . ( F . ( G ) cnk mutant showing a wing-mirror-image duplication (arrowheads). ( H ) Distribution of Wg expression and MAPK activity in early third instar wing discs. MAPK activity, detected with an anti-dpERK antibody (green), is excluded from the wing pouch (arrowhead) and complementary to Wg expression (red).

    Techniques Used: Transformation Assay, Expressing, Dominant Negative Mutation, Over Expression, Clone Assay, Generated, Mutagenesis, Activity Assay

    10) Product Images from "Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation"

    Article Title: Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation

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

    doi: 10.1073/pnas.1834876100

    Activation of MAPK. Total cell lysates (10 μg) were subjected to immunoblot analysis with anti-phospho-MAPK antibody ( Upper ) or anti-MAPK antibody ( Lower ). Lane 1, TIG-1 infected with a retroviral vector expressing hTERT (T); lane 2, TIG-1 infected with retroviral vector expressing T, SV40 ER (S), and H-RasV12 (R); lane 3, TIG-3 infected with a retroviral vector expressing T; lane 4, TIG-3 infected with retroviral vectors expressing T, S, and R; lane 5, BJ infected with a retroviral vector expressing T; lane 6, BJ infected with retroviral vectors expressing T, S, and R; lane 7, IMR-90 infected with a retroviral vector expressing T; lane 8, IMR-90 infected with retroviral vectors expressing T, S, and R; lane 9, REF; lane 10, REF infected with retroviral vectors expressing S and R.
    Figure Legend Snippet: Activation of MAPK. Total cell lysates (10 μg) were subjected to immunoblot analysis with anti-phospho-MAPK antibody ( Upper ) or anti-MAPK antibody ( Lower ). Lane 1, TIG-1 infected with a retroviral vector expressing hTERT (T); lane 2, TIG-1 infected with retroviral vector expressing T, SV40 ER (S), and H-RasV12 (R); lane 3, TIG-3 infected with a retroviral vector expressing T; lane 4, TIG-3 infected with retroviral vectors expressing T, S, and R; lane 5, BJ infected with a retroviral vector expressing T; lane 6, BJ infected with retroviral vectors expressing T, S, and R; lane 7, IMR-90 infected with a retroviral vector expressing T; lane 8, IMR-90 infected with retroviral vectors expressing T, S, and R; lane 9, REF; lane 10, REF infected with retroviral vectors expressing S and R.

    Techniques Used: Activation Assay, Infection, Plasmid Preparation, Expressing

    11) Product Images from "Multiple strategies of oxygen supply in Drosophila malignancies identify tracheogenesis as a novel cancer hallmark"

    Article Title: Multiple strategies of oxygen supply in Drosophila malignancies identify tracheogenesis as a novel cancer hallmark

    Journal: Scientific Reports

    doi: 10.1038/srep09061

    Several cancer-related pathways are implicated in the tumourigenic process triggered by the MEN-lgl KD system. (a–g) Imaginal wing discs from w ; Rpl27A 1 , en-Gal4 , UAS-GFP /+; UAS-lgl-RNAi /+ individuals. (a) The overall disc structure is shown by aPKC staining; the posterior (P), GFP-positive compartment is visibly altered and the section along the z axis shows loss of apical-basal cell polarity. A: apical; B: basal. Ectopic expression of pJNK is shown in red. (b) Yki accumulation is visible in the P compartment (magnification). (c–g) Ectopic expression of dMyc, dIAP1, pAKT, dpERK and MMP1 respectively is evident in the P compartment. (h) Imaginal wing discs from w, UAS-bsk DN ; Rpl27A 1 , en-Gal4 , UAS-GFP /+; UAS-lgl-RNAi /+ individuals. As can be seen, disc structure, growth and invasiveness (MMP1 staining, red) are almost entirely rescued. The A/P border is dotted in green and disc contour is outlined in white. The genotype of the P compartment is indicated in each image.
    Figure Legend Snippet: Several cancer-related pathways are implicated in the tumourigenic process triggered by the MEN-lgl KD system. (a–g) Imaginal wing discs from w ; Rpl27A 1 , en-Gal4 , UAS-GFP /+; UAS-lgl-RNAi /+ individuals. (a) The overall disc structure is shown by aPKC staining; the posterior (P), GFP-positive compartment is visibly altered and the section along the z axis shows loss of apical-basal cell polarity. A: apical; B: basal. Ectopic expression of pJNK is shown in red. (b) Yki accumulation is visible in the P compartment (magnification). (c–g) Ectopic expression of dMyc, dIAP1, pAKT, dpERK and MMP1 respectively is evident in the P compartment. (h) Imaginal wing discs from w, UAS-bsk DN ; Rpl27A 1 , en-Gal4 , UAS-GFP /+; UAS-lgl-RNAi /+ individuals. As can be seen, disc structure, growth and invasiveness (MMP1 staining, red) are almost entirely rescued. The A/P border is dotted in green and disc contour is outlined in white. The genotype of the P compartment is indicated in each image.

    Techniques Used: Staining, Expressing

    12) Product Images from "Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish"

    Article Title: Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish

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

    doi: 10.1073/pnas.0611302104

    Human kRASG12D transgene expression can be induced by Cre-mediated recombination in stable transgenic B-actin-LoxP-EGFP-LoxP-kRASG12D zebrafish. ( A ) Diagram of B-actin-LoxP-EGFP-LoxP-kRASG12D transgene. ( B–E ) Stable transgenic animals (line 25A) at 24 hpf ( B and C ), and 44 dpf ( D and E ). ( B and D ) Bright-field. ( C and E ) EGFP fluorescence. ( F and G ) Whole-mount in situ hybridization for kRASG12D performed on 24 hpf double transgenic embryos ( LGL-RAS ; hsp70-Cre ) with heat shock ( F ) or without ( G ). Arrowheads denote kRASG12D transcript-expressing cells. ( H and I ) Whole-mount immunostaining with anti-phospho-ERK1/2 antibody performed on 24 hpf double transgenic embryos with heat shock ( H ) or without ( I ). Cells with ERK1/2 activation are denoted by arrows. ( J ) Percentage of recombination at the genomic DNA in single embryos heat shocked from 4 to 5 hpf and analyzed at 8, 16, and 24 hpf. ( K ) Number of kRASG12D -expressing cells in single embryos heat shocked from 4 to 5 hpf and analyzed at 8, 12, 16, 20, and 24 hpf. Statistic significance ( P
    Figure Legend Snippet: Human kRASG12D transgene expression can be induced by Cre-mediated recombination in stable transgenic B-actin-LoxP-EGFP-LoxP-kRASG12D zebrafish. ( A ) Diagram of B-actin-LoxP-EGFP-LoxP-kRASG12D transgene. ( B–E ) Stable transgenic animals (line 25A) at 24 hpf ( B and C ), and 44 dpf ( D and E ). ( B and D ) Bright-field. ( C and E ) EGFP fluorescence. ( F and G ) Whole-mount in situ hybridization for kRASG12D performed on 24 hpf double transgenic embryos ( LGL-RAS ; hsp70-Cre ) with heat shock ( F ) or without ( G ). Arrowheads denote kRASG12D transcript-expressing cells. ( H and I ) Whole-mount immunostaining with anti-phospho-ERK1/2 antibody performed on 24 hpf double transgenic embryos with heat shock ( H ) or without ( I ). Cells with ERK1/2 activation are denoted by arrows. ( J ) Percentage of recombination at the genomic DNA in single embryos heat shocked from 4 to 5 hpf and analyzed at 8, 16, and 24 hpf. ( K ) Number of kRASG12D -expressing cells in single embryos heat shocked from 4 to 5 hpf and analyzed at 8, 12, 16, 20, and 24 hpf. Statistic significance ( P

    Techniques Used: Expressing, Transgenic Assay, Fluorescence, In Situ Hybridization, Immunostaining, Activation Assay

    13) Product Images from "Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses"

    Article Title: Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2018.05.002

    NK1 Treatment Restores FGF2 Activity in Fibroblasts from MPS I- and MPS IIIB-Affected Patients Titration of FGF receptor activation was performed by stimulating for 10 min starved MPS I and MPS IIIB fibroblasts with increasing doses of FGF2, both in the absence and in the presence of 10 −6 M NK1, and evaluating the phosphorylation levels of ERK1/2 by western blotting. The upper blots were stripped and re-probed with anti-ERK1/2 antibody. Anti-γ-tubulin antibody was used to ensure equal loading of proteins in all lanes. The blots reported are representative of three independent experiments of equal design.
    Figure Legend Snippet: NK1 Treatment Restores FGF2 Activity in Fibroblasts from MPS I- and MPS IIIB-Affected Patients Titration of FGF receptor activation was performed by stimulating for 10 min starved MPS I and MPS IIIB fibroblasts with increasing doses of FGF2, both in the absence and in the presence of 10 −6 M NK1, and evaluating the phosphorylation levels of ERK1/2 by western blotting. The upper blots were stripped and re-probed with anti-ERK1/2 antibody. Anti-γ-tubulin antibody was used to ensure equal loading of proteins in all lanes. The blots reported are representative of three independent experiments of equal design.

    Techniques Used: Activity Assay, Titration, Activation Assay, Western Blot

    14) Product Images from "The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development"

    Article Title: The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development

    Journal: EvoDevo

    doi: 10.1186/2041-9139-4-10

    Expression of the ERK MAP kinase gene rolled ( rl ) and distribution of activated ERK MAP kinase during oviparous and viviparous oogenesis. A-D . rl mRNA is detected in the trophocytes of the germaria and in young oocytes during both oviparous ( A,B ) and viviparous ( C,D ) oogenesis. E-M . Activated ERK MAP kinase (dpERK) is first found in early oocytes prior to being extruded from germarium during both oviparous ( E and M , see inset for close-up) and viviparous ( I,J ) development. Following extrusion, dpERK is distributed throughout the oocyte in oviparous ( F,G ) and viviparous ( K,L ) cases. During late previtellogenesis of oviparous oocytes, however, dpERK becomes restricted to the most posterior region of the oocyte ( H , arrowhead). The progressive nature of this restriction occurs as the oocyte increases in size ( M , arrow indicates younger to older oocytes). g, germarium; o, oocyte. Scale bars: 100 μm for A,B ; 20 μm for C,D ; 100 μm in E-H; 20 μm for I-L; 100 μm in M,N (20 μm in inset). Staging according to [ 8 ].
    Figure Legend Snippet: Expression of the ERK MAP kinase gene rolled ( rl ) and distribution of activated ERK MAP kinase during oviparous and viviparous oogenesis. A-D . rl mRNA is detected in the trophocytes of the germaria and in young oocytes during both oviparous ( A,B ) and viviparous ( C,D ) oogenesis. E-M . Activated ERK MAP kinase (dpERK) is first found in early oocytes prior to being extruded from germarium during both oviparous ( E and M , see inset for close-up) and viviparous ( I,J ) development. Following extrusion, dpERK is distributed throughout the oocyte in oviparous ( F,G ) and viviparous ( K,L ) cases. During late previtellogenesis of oviparous oocytes, however, dpERK becomes restricted to the most posterior region of the oocyte ( H , arrowhead). The progressive nature of this restriction occurs as the oocyte increases in size ( M , arrow indicates younger to older oocytes). g, germarium; o, oocyte. Scale bars: 100 μm for A,B ; 20 μm for C,D ; 100 μm in E-H; 20 μm for I-L; 100 μm in M,N (20 μm in inset). Staging according to [ 8 ].

    Techniques Used: Expressing

    15) Product Images from "Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses"

    Article Title: Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2018.05.002

    NK1 Treatment Restores FGF2 Activity in Fibroblasts from MPS I- and MPS IIIB-Affected Patients Titration of FGF receptor activation was performed by stimulating for 10 min starved MPS I and MPS IIIB fibroblasts with increasing doses of FGF2, both in the absence and in the presence of 10 −6 M NK1, and evaluating the phosphorylation levels of ERK1/2 by western blotting. The upper blots were stripped and re-probed with anti-ERK1/2 antibody. Anti-γ-tubulin antibody was used to ensure equal loading of proteins in all lanes. The blots reported are representative of three independent experiments of equal design.
    Figure Legend Snippet: NK1 Treatment Restores FGF2 Activity in Fibroblasts from MPS I- and MPS IIIB-Affected Patients Titration of FGF receptor activation was performed by stimulating for 10 min starved MPS I and MPS IIIB fibroblasts with increasing doses of FGF2, both in the absence and in the presence of 10 −6 M NK1, and evaluating the phosphorylation levels of ERK1/2 by western blotting. The upper blots were stripped and re-probed with anti-ERK1/2 antibody. Anti-γ-tubulin antibody was used to ensure equal loading of proteins in all lanes. The blots reported are representative of three independent experiments of equal design.

    Techniques Used: Activity Assay, Titration, Activation Assay, Western Blot

    16) Product Images from "Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6"

    Article Title: Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

    Journal: Endocrinology

    doi: 10.1210/en.2013-1822

    Activation of MAPK in endometrial cells treated with lipopeptide PAMPs. Endometrial epithelial cells (A) and stromal cells (B) were collected 0, 5, 10, 15, 20, or 25 minutes after treatment with 100 ng/mL PAM or 100 ng/mL FSL-1. The protein from the cells was analyzed by SDS-PAGE and immunoblotted with antibodies against total and phosphorylated forms of p38 (t-p38 and p-p38) and ERK1/2 (tERK1/2 and pERK1/2; □, tERK2; ■, pERK2), and α-tubulin as visual confirmation of the precision of protein loading and transfer. The image for each cell type is representative of 3 independent experiments for PAM (left panel) or FSL-1 (right panel), and the histograms represent the mean ± SEM of densitometric analysis of the ratio of phosphorylated p-p38 to t-p38, pERK1 to tERK1 or pERK2 to tERK2, expressed as fold activation compared with time 0. Values differ from time 0 when data were analyzed by ANOVA, using the Dunnett pairwise multiple comparison t test: *, P
    Figure Legend Snippet: Activation of MAPK in endometrial cells treated with lipopeptide PAMPs. Endometrial epithelial cells (A) and stromal cells (B) were collected 0, 5, 10, 15, 20, or 25 minutes after treatment with 100 ng/mL PAM or 100 ng/mL FSL-1. The protein from the cells was analyzed by SDS-PAGE and immunoblotted with antibodies against total and phosphorylated forms of p38 (t-p38 and p-p38) and ERK1/2 (tERK1/2 and pERK1/2; □, tERK2; ■, pERK2), and α-tubulin as visual confirmation of the precision of protein loading and transfer. The image for each cell type is representative of 3 independent experiments for PAM (left panel) or FSL-1 (right panel), and the histograms represent the mean ± SEM of densitometric analysis of the ratio of phosphorylated p-p38 to t-p38, pERK1 to tERK1 or pERK2 to tERK2, expressed as fold activation compared with time 0. Values differ from time 0 when data were analyzed by ANOVA, using the Dunnett pairwise multiple comparison t test: *, P

    Techniques Used: Activation Assay, SDS Page

    Attenuation of endometrial cell responses to lipopeptides by inhibition of MAPK. Endometrial epithelial (A and B) or stromal cells (C and D) were treated for 30 minutes in medium containing vehicle (V), ERK1/2 inhibitor (ERKi) (ERK activation inhibitor peptide I, 10 μM) or p38 inhibitor (p38i) (InSolution SB 203580, 10 μM) and then were cultured in the same treatment for 6 hours in control medium or medium containing 100 ng/mL PAM (A and C) or 100 ng/mL FSL-1 (B and D). Supernatants were harvested to measure the accumulation of IL-6 by ELISA, and results are expressed as a percentage of treatment with PAM (A and C) or FSL-1 (B and D). Data are presented as mean + SEM percentages and represent 3 independent experiments. Values differ from those for PAMP when data were analyzed by ANOVA using the Dunnett pairwise multiple comparison t test: *, P
    Figure Legend Snippet: Attenuation of endometrial cell responses to lipopeptides by inhibition of MAPK. Endometrial epithelial (A and B) or stromal cells (C and D) were treated for 30 minutes in medium containing vehicle (V), ERK1/2 inhibitor (ERKi) (ERK activation inhibitor peptide I, 10 μM) or p38 inhibitor (p38i) (InSolution SB 203580, 10 μM) and then were cultured in the same treatment for 6 hours in control medium or medium containing 100 ng/mL PAM (A and C) or 100 ng/mL FSL-1 (B and D). Supernatants were harvested to measure the accumulation of IL-6 by ELISA, and results are expressed as a percentage of treatment with PAM (A and C) or FSL-1 (B and D). Data are presented as mean + SEM percentages and represent 3 independent experiments. Values differ from those for PAMP when data were analyzed by ANOVA using the Dunnett pairwise multiple comparison t test: *, P

    Techniques Used: Inhibition, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection"

    Article Title: Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12756

    The effect of the TG2 inhibitor Z-DON on PMA and forskolin-induced ERK1/2 activation. (A) H9c2 cells were pretreated for 1 h with the TG2 inhibitor Z-DON (150 μM) prior to 5 min stimulation with PMA (1 μM) or forskolin (10 μM).
    Figure Legend Snippet: The effect of the TG2 inhibitor Z-DON on PMA and forskolin-induced ERK1/2 activation. (A) H9c2 cells were pretreated for 1 h with the TG2 inhibitor Z-DON (150 μM) prior to 5 min stimulation with PMA (1 μM) or forskolin (10 μM).

    Techniques Used: Activation Assay

    The effect of the TG2 inhibitor R283 on PMA- and forskolin-induced ERK1/2 activation. (A) H9c2 cells were pretreated for 1 h with the TG2 inhibitor R283 (200 μM) prior to 5 min stimulation with PMA (1 μM) or forskolin (10 μM).
    Figure Legend Snippet: The effect of the TG2 inhibitor R283 on PMA- and forskolin-induced ERK1/2 activation. (A) H9c2 cells were pretreated for 1 h with the TG2 inhibitor R283 (200 μM) prior to 5 min stimulation with PMA (1 μM) or forskolin (10 μM).

    Techniques Used: Activation Assay

    18) Product Images from "B-Cell Receptor- and Phorbol Ester-Induced NF-?B and c-Jun N-Terminal Kinase Activation in B Cells Requires Novel Protein Kinase C's"

    Article Title: B-Cell Receptor- and Phorbol Ester-Induced NF-?B and c-Jun N-Terminal Kinase Activation in B Cells Requires Novel Protein Kinase C's

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.19.6640-6650.2001

    (A) IKKγ expression rescues LPS- and IL-1β- but not PMA-induced NF-κB activation of 1.3E2 cells. 70Z/3 cells, three 1.3E2 IKKγ-expressing clones, and 1.3E2 cells were treated with PMA, LPS, or IL-1β for 30 min and NF-κB DNA-binding activity was determined by EMSA. IKKγ protein was determined by Western blotting (lower panel). (B and C) Activation of IKK and JNK1 in response to PMA is defective in 1.3E2 and 1.3E2 IKKγ cells. (B) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), and extracts were analyzed for IKKα protein expression. IKK activity was determined in an in vitro kinase reaction after IKK immunoprecipitation with an anti-IKKα antibody. GstIκBα1–53 was used as a substrate. (C) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), LPS (20 min), or UV light (20 min) and cellular extracts were analyzed for JNK1 expression (upper panel). The hyperphosphorylated 46-kDa JNK1 isoform is indicated by a star. JNK1 kinase activity was determined in an in vitro kinase reaction after immunoprecipitation of JNK1. Kinase activity was determined with recombinant GstJun1–79 as substrate. (D) Activation of ERK1/2 is not defective in 1.3E2 cells. Cells were stimulated with PMA for 10 min and cellular extracts were analyzed by Western blotting using JNK1, phospho-ERK1/2, or ERK1/2 antibodies (as indicated). Migration of the hyperphosphorylated 46-kDa JNK1 is indicated by a star.
    Figure Legend Snippet: (A) IKKγ expression rescues LPS- and IL-1β- but not PMA-induced NF-κB activation of 1.3E2 cells. 70Z/3 cells, three 1.3E2 IKKγ-expressing clones, and 1.3E2 cells were treated with PMA, LPS, or IL-1β for 30 min and NF-κB DNA-binding activity was determined by EMSA. IKKγ protein was determined by Western blotting (lower panel). (B and C) Activation of IKK and JNK1 in response to PMA is defective in 1.3E2 and 1.3E2 IKKγ cells. (B) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), and extracts were analyzed for IKKα protein expression. IKK activity was determined in an in vitro kinase reaction after IKK immunoprecipitation with an anti-IKKα antibody. GstIκBα1–53 was used as a substrate. (C) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), LPS (20 min), or UV light (20 min) and cellular extracts were analyzed for JNK1 expression (upper panel). The hyperphosphorylated 46-kDa JNK1 isoform is indicated by a star. JNK1 kinase activity was determined in an in vitro kinase reaction after immunoprecipitation of JNK1. Kinase activity was determined with recombinant GstJun1–79 as substrate. (D) Activation of ERK1/2 is not defective in 1.3E2 cells. Cells were stimulated with PMA for 10 min and cellular extracts were analyzed by Western blotting using JNK1, phospho-ERK1/2, or ERK1/2 antibodies (as indicated). Migration of the hyperphosphorylated 46-kDa JNK1 is indicated by a star.

    Techniques Used: Expressing, Activation Assay, Clone Assay, Binding Assay, Activity Assay, Western Blot, In Vitro, Immunoprecipitation, Recombinant, Migration

    19) Product Images from "L-Carnitine rescues ketamine-induced attenuated heart rate and MAPK (ERK) activity in zebrafish embryos"

    Article Title: L-Carnitine rescues ketamine-induced attenuated heart rate and MAPK (ERK) activity in zebrafish embryos

    Journal: Reproductive toxicology (Elmsford, N.Y.)

    doi: 10.1016/j.reprotox.2011.10.004

    Schematic presentation of a potential mechanism of ERK/MAPK modulation by ketamine and L-carnitine. NMDA receptors are Ca 2+ permeable. In presence of ketamine, this property of the NMDA receptor is lost. NMDA receptor permeable Ca 2+ is known to couple
    Figure Legend Snippet: Schematic presentation of a potential mechanism of ERK/MAPK modulation by ketamine and L-carnitine. NMDA receptors are Ca 2+ permeable. In presence of ketamine, this property of the NMDA receptor is lost. NMDA receptor permeable Ca 2+ is known to couple

    Techniques Used:

    20) Product Images from "Pirfenidone anti-fibrotic effects are partially mediated by the inhibition of MUC1 bioactivation"

    Article Title: Pirfenidone anti-fibrotic effects are partially mediated by the inhibition of MUC1 bioactivation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27526

    Pirfenidone (PFD) inhibits the TGF-β1-induced β-catenin activation but not the SMAD3 and ERK1/2 phosphorylation. A549 ( A ) and MRC5 ( B ) cells were stimulated 40 min with TGFβ1 5 ng/ml in the presence or absence of PFD 50 µM. Total protein was analyzed by western blot and quantified by densitometry. Protein expression of phospho (p)-SMAD3, p-ERK1/2 and active (act)-β-catenin was measured. Data are expressed as the ratio to total Smad3, total ERK1/2 or total β-catenin protein. Sample Western blots from a single representative experiment are shown. One-way ANOVA was followed by the post hoc Bonferroni test. * P
    Figure Legend Snippet: Pirfenidone (PFD) inhibits the TGF-β1-induced β-catenin activation but not the SMAD3 and ERK1/2 phosphorylation. A549 ( A ) and MRC5 ( B ) cells were stimulated 40 min with TGFβ1 5 ng/ml in the presence or absence of PFD 50 µM. Total protein was analyzed by western blot and quantified by densitometry. Protein expression of phospho (p)-SMAD3, p-ERK1/2 and active (act)-β-catenin was measured. Data are expressed as the ratio to total Smad3, total ERK1/2 or total β-catenin protein. Sample Western blots from a single representative experiment are shown. One-way ANOVA was followed by the post hoc Bonferroni test. * P

    Techniques Used: Activation Assay, Western Blot, Expressing

    21) Product Images from "A GNAS Mutation Found in Pancreatic Intraductal Papillary Mucinous Neoplasms Induces Drastic Alterations of Gene Expression Profiles with Upregulation of Mucin Genes"

    Article Title: A GNAS Mutation Found in Pancreatic Intraductal Papillary Mucinous Neoplasms Induces Drastic Alterations of Gene Expression Profiles with Upregulation of Mucin Genes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087875

    MAPK activity contributes to expression of mucin genes under different state of G protein activity. (A) Immunoblots of total lysates of cells transfected with the empty vector (Vec), wild-type GNAS-V5 (GW), and mutated GNAS-V5 (R201H; abbreviated as GM) with or without U0126, a potent mitogen-activated protein kinase kinase (MAP2K) inhibitor. (B) Cyclic AMP quantified using an enzyme immunoassay. U0126 treatment did not affect cAMP levels in PK-8 cells but downregulated cAMP in PCI-35 cells, except in the mutated GNAS transfectant. (C and D) A quantitative real-time PCR assay. (C) MUC2 was consistently downregulated by U0126 in PK-8 and PCI-35 cells, regardless of the presence of exogenous GNAS . (D) MUC5AC was consistently downregulated in PCI-35 cells, regardless of the presence of exogenous GNAS , and upregulated by U0126 in PK-8 cells expressing exogenous mutated GNAS. Values obtained from independently duplicated experiments were plotted. Error bars indicate standard error. *p
    Figure Legend Snippet: MAPK activity contributes to expression of mucin genes under different state of G protein activity. (A) Immunoblots of total lysates of cells transfected with the empty vector (Vec), wild-type GNAS-V5 (GW), and mutated GNAS-V5 (R201H; abbreviated as GM) with or without U0126, a potent mitogen-activated protein kinase kinase (MAP2K) inhibitor. (B) Cyclic AMP quantified using an enzyme immunoassay. U0126 treatment did not affect cAMP levels in PK-8 cells but downregulated cAMP in PCI-35 cells, except in the mutated GNAS transfectant. (C and D) A quantitative real-time PCR assay. (C) MUC2 was consistently downregulated by U0126 in PK-8 and PCI-35 cells, regardless of the presence of exogenous GNAS . (D) MUC5AC was consistently downregulated in PCI-35 cells, regardless of the presence of exogenous GNAS , and upregulated by U0126 in PK-8 cells expressing exogenous mutated GNAS. Values obtained from independently duplicated experiments were plotted. Error bars indicate standard error. *p

    Techniques Used: Activity Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    22) Product Images from "Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway"

    Article Title: Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway

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

    doi: 10.1073/pnas.241507698

    Changes in ERK-, p38-, and NR2-subunit tyrosine phosphorylation in brain of female rats in proestrus and diestrus. Brain tissues from female rats in diestrus or proestrus were homogenized in Tris-acetate buffer (100 mM, pH 7.4) containing 0.1 mM EGTA and processed for Western blotting. ( A ) Membranes were processed with monoclonal antibodies against total (top half) or phosphorylated ERK (bottom half). ( B and C ) Blots similar to those shown in A were quantified, and results for ERK1 (hatched bars) and ERK2 (black bars) were expressed as percentages of the values found in samples from females in diestrus (means ± SEM of 6–8 animals). *, P
    Figure Legend Snippet: Changes in ERK-, p38-, and NR2-subunit tyrosine phosphorylation in brain of female rats in proestrus and diestrus. Brain tissues from female rats in diestrus or proestrus were homogenized in Tris-acetate buffer (100 mM, pH 7.4) containing 0.1 mM EGTA and processed for Western blotting. ( A ) Membranes were processed with monoclonal antibodies against total (top half) or phosphorylated ERK (bottom half). ( B and C ) Blots similar to those shown in A were quantified, and results for ERK1 (hatched bars) and ERK2 (black bars) were expressed as percentages of the values found in samples from females in diestrus (means ± SEM of 6–8 animals). *, P

    Techniques Used: Western Blot

    23) Product Images from "Phosphatase-defective LEOPARD syndrome mutations in PTPN11 gene have gain-of-function effects during Drosophila development"

    Article Title: Phosphatase-defective LEOPARD syndrome mutations in PTPN11 gene have gain-of-function effects during Drosophila development

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddn336

    MAPK activation in ubiquitous LEOPARD and Noonan syndrome-transgene expressing larvae. Immunoblots of protein lysates from second instar larvae using antibodies against dpERK (activated MAPK) and ERK (all forms of MAPK). ERK is expressed at approximately equal levels in all fly lines. The expression of dpERK is increased in the wild-type and mutant transgenic larvae, as compared to the wild-type larvae showing a minimal level of dpERK.
    Figure Legend Snippet: MAPK activation in ubiquitous LEOPARD and Noonan syndrome-transgene expressing larvae. Immunoblots of protein lysates from second instar larvae using antibodies against dpERK (activated MAPK) and ERK (all forms of MAPK). ERK is expressed at approximately equal levels in all fly lines. The expression of dpERK is increased in the wild-type and mutant transgenic larvae, as compared to the wild-type larvae showing a minimal level of dpERK.

    Techniques Used: Activation Assay, Expressing, Western Blot, Mutagenesis, Transgenic Assay

    24) Product Images from "The Role of Identified Neurotransmitter Systems in the Response of Insular Cortex to Unfamiliar Taste: Activation of ERK1–2 and Formation of a Memory Trace"

    Article Title: The Role of Identified Neurotransmitter Systems in the Response of Insular Cortex to Unfamiliar Taste: Activation of ERK1–2 and Formation of a Memory Trace

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.20-18-07017.2000

    Effect of various ligands microinjected into the IC on ERK1–2 activation. A , Effect on taste-induced ERK1–2 activation. Top , representative blots of activated ( dpERK1–2 ) and total ERK1–2 ( ERK ) from animals microinjected with the different neurotransmitter ligands 20 min before exposure to saccharin ( black and gray bars ) and water ( white bar ). Bottom , Quantification of the results from the blots ( n = 10 per group). The level of dpERK1–2 in animals microinjected with ACSF and exposed to saccharin ( black bar ) is used as standard. Ratio of activation is presented as dpERK experimental/dpERK saccharin. B , Effect of the ligands on the basal level of ERK1–2 activation. Top , Representative blots of dpERK1–2 and ERK1–2 from animals microinjected with the ligands and exposed to water 20 min later. Bottom , Quantification of the results from the blots ( n = 12). The level of dpERK1–2 in animals microinjected with ACSF and exposed to water ( black bar ) is used as standard. Ratio of activation is presented as dpERK experimental/dpERK water. W , Water; S, saccharin.
    Figure Legend Snippet: Effect of various ligands microinjected into the IC on ERK1–2 activation. A , Effect on taste-induced ERK1–2 activation. Top , representative blots of activated ( dpERK1–2 ) and total ERK1–2 ( ERK ) from animals microinjected with the different neurotransmitter ligands 20 min before exposure to saccharin ( black and gray bars ) and water ( white bar ). Bottom , Quantification of the results from the blots ( n = 10 per group). The level of dpERK1–2 in animals microinjected with ACSF and exposed to saccharin ( black bar ) is used as standard. Ratio of activation is presented as dpERK experimental/dpERK saccharin. B , Effect of the ligands on the basal level of ERK1–2 activation. Top , Representative blots of dpERK1–2 and ERK1–2 from animals microinjected with the ligands and exposed to water 20 min later. Bottom , Quantification of the results from the blots ( n = 12). The level of dpERK1–2 in animals microinjected with ACSF and exposed to water ( black bar ) is used as standard. Ratio of activation is presented as dpERK experimental/dpERK water. W , Water; S, saccharin.

    Techniques Used: Activation Assay

    25) Product Images from "cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells"

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-018-0100-0

    SM83 treatment results in anti-tumor and anti-metastasis effect. a Lungs of NOD/SCID mice bearing MDA-MB231 tumors were collected 2 weeks after the last injection with SM83 (upper panel, see Fig. S 4a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n = 8; sum of two independent experiments shown in Fig. S 4a–b ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a
    Figure Legend Snippet: SM83 treatment results in anti-tumor and anti-metastasis effect. a Lungs of NOD/SCID mice bearing MDA-MB231 tumors were collected 2 weeks after the last injection with SM83 (upper panel, see Fig. S 4a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n = 8; sum of two independent experiments shown in Fig. S 4a–b ; * P = 0.0238. Unpaired two-tailed t- test) and c size (35 metastases/group; * P = 0.0107. Unpaired two-tailed t- test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a

    Techniques Used: Mouse Assay, Multiple Displacement Amplification, Injection, Staining, Two Tailed Test, Western Blot

    IAP inhibition hinders EGFR signaling independently from the receptor downregulation. a SUM159 and b MCF10A cells were serum-starved, pre-treated or not with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blots were performed to evaluate the total and activated levels of EGFR and ERK1/2, and total levels of Snai2. cIAP1 is shown to control the efficiency of the treatment. c MCF10A cell viability was tested by CellTiter-Glo 24 h after treatment with 100 nM SM83. d BT549 cells ectopically expressing Myc/Flag-tagged EGFR, silenced with control and cIAP1-specific siRNAs, were serum-starved overnight and then stimulated with 20 ng/ml EGF. Western blot was performed to detect the total levels of ectopic EGFR (Myc), cIAP1, and Snai2. e BT549 cells ectopically expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated or not 1 h with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blot was performed to detect the total levels of ectopic EGFR (Myc), ERK1/2, cIAP1, and Snai2, and the activated form of ERK1/2. f Tumors described in Fig. 1b were analyzed by western blot to detect the activation of ERK1/2, and the total levels of EGFR and ERK1/2
    Figure Legend Snippet: IAP inhibition hinders EGFR signaling independently from the receptor downregulation. a SUM159 and b MCF10A cells were serum-starved, pre-treated or not with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blots were performed to evaluate the total and activated levels of EGFR and ERK1/2, and total levels of Snai2. cIAP1 is shown to control the efficiency of the treatment. c MCF10A cell viability was tested by CellTiter-Glo 24 h after treatment with 100 nM SM83. d BT549 cells ectopically expressing Myc/Flag-tagged EGFR, silenced with control and cIAP1-specific siRNAs, were serum-starved overnight and then stimulated with 20 ng/ml EGF. Western blot was performed to detect the total levels of ectopic EGFR (Myc), cIAP1, and Snai2. e BT549 cells ectopically expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated or not 1 h with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blot was performed to detect the total levels of ectopic EGFR (Myc), ERK1/2, cIAP1, and Snai2, and the activated form of ERK1/2. f Tumors described in Fig. 1b were analyzed by western blot to detect the activation of ERK1/2, and the total levels of EGFR and ERK1/2

    Techniques Used: Inhibition, Western Blot, Expressing, Activation Assay

    Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test
    Figure Legend Snippet: Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: * P = 0.0151, ** P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 * P = 0.0290, EGF 3 h siCtr vs. sicIAP1 * P = 0.0330; n = 5; two-tailed paired t- test

    Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c A panel of breast cancer cell lines was tested to compare the levels of Snai2. BaA basal “A”, BaB basal “B”, Lu Luminal [ 55 ]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels
    Figure Legend Snippet: EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c A panel of breast cancer cell lines was tested to compare the levels of Snai2. BaA basal “A”, BaB basal “B”, Lu Luminal [ 55 ]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 ( f ) and BT549 ( g ) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Transfection, RNA Sequencing Assay

    26) Product Images from "Protein O-GlcNAcylation Is Required for Fibroblast Growth Factor Signaling in Drosophila"

    Article Title: Protein O-GlcNAcylation Is Required for Fibroblast Growth Factor Signaling in Drosophila

    Journal: Science signaling

    doi: 10.1126/scisignal.2002335

    Epistatic relationship between nst and htl . ( A ) Expression of UAS::Nst rescues nst MZ mutant cells autonomously. Embryos of the indicated genotypes were stained with antibodies recognizing either Eve (stage 11, top row) or Verm (stage 15, bottom row). ( B ) Quantification of Eve-positive hemisegments in embryos of indicated genotypes ( n ). ( C ) Percentage of embryos with either intact dorsal trunk (DT) or the presence (Verm+) or absence (Verm−) of Verm staining ( n = 100 embryos) for the indicated genotypes. The mutase-dead Nst[S68A] mutant served as a control. ( D ) Expression of constitutively active Htl (λHtl) in the mesoderm of nst mutant embryos stained with antibodies recognizing Eve. Arrows mark enlarged Eve cell clusters caused by expression of λHtl in maternal nst ( nst M ) mutants, which are lacking in the nst MZ mutants expressing λHtl. ( E ) Quantification of the Eve-positive hemisegments of embryos expressing λHtl in nst MZ mutants ( n ). ( F ) MAPK activation in the mesoderm was detected with antibodies against dpERK (red) in stage 8 embryos with λHtl in maternal nst M mutants (top row) and nst MZ mutants expressing λHtl (bottom row). The mesoderm nuclei were stained with antibodies recognizing Twi (green) and the presence of the paternal balancer chromosome by anti-βGal (green). twi > > Nst: twi::Gal4,UAS::Nst; btl > > Nst: btl::Gal4,UAS::Nst; twi > > NstS68A: twi::Gal4,UAS::Nst [S86A]; btl > > NstS68A: btl::Gal4, UAS:: Nst [S86A]; twi > > λ Htl: twi::Gal4,UAS:: λ Htl .
    Figure Legend Snippet: Epistatic relationship between nst and htl . ( A ) Expression of UAS::Nst rescues nst MZ mutant cells autonomously. Embryos of the indicated genotypes were stained with antibodies recognizing either Eve (stage 11, top row) or Verm (stage 15, bottom row). ( B ) Quantification of Eve-positive hemisegments in embryos of indicated genotypes ( n ). ( C ) Percentage of embryos with either intact dorsal trunk (DT) or the presence (Verm+) or absence (Verm−) of Verm staining ( n = 100 embryos) for the indicated genotypes. The mutase-dead Nst[S68A] mutant served as a control. ( D ) Expression of constitutively active Htl (λHtl) in the mesoderm of nst mutant embryos stained with antibodies recognizing Eve. Arrows mark enlarged Eve cell clusters caused by expression of λHtl in maternal nst ( nst M ) mutants, which are lacking in the nst MZ mutants expressing λHtl. ( E ) Quantification of the Eve-positive hemisegments of embryos expressing λHtl in nst MZ mutants ( n ). ( F ) MAPK activation in the mesoderm was detected with antibodies against dpERK (red) in stage 8 embryos with λHtl in maternal nst M mutants (top row) and nst MZ mutants expressing λHtl (bottom row). The mesoderm nuclei were stained with antibodies recognizing Twi (green) and the presence of the paternal balancer chromosome by anti-βGal (green). twi > > Nst: twi::Gal4,UAS::Nst; btl > > Nst: btl::Gal4,UAS::Nst; twi > > NstS68A: twi::Gal4,UAS::Nst [S86A]; btl > > NstS68A: btl::Gal4, UAS:: Nst [S86A]; twi > > λ Htl: twi::Gal4,UAS:: λ Htl .

    Techniques Used: Expressing, Mutagenesis, Staining, Activation Assay

    27) Product Images from "Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells"

    Article Title: Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086865

    Growth factor-induced myofibroblast differentiation is reversible. (A) Phase images are shown of ADSCs treated with TGF-β (TGF-β, 4d) or with bFGF (bFGF, 4d) for 4 days (left). SSFM containing bFGF was added to TGF-β-differentiated cells and TGF-β was added to bFGF-differentiated cells. Phase images were captured after 4 days (right). Images are representative of more than three independent experiments. Scale bar = 50 µm. (B) Lysates were prepared from cells grown under the six indicated conditions and were used for immunoblots with anti-α-SMA monoclonal antibody. GAPDH was used as the loading control. Blot is representative of three independent experiments. Legend indicates cell treatments in lanes 1–6. Unt., 4d = untreated for 4 days.
    Figure Legend Snippet: Growth factor-induced myofibroblast differentiation is reversible. (A) Phase images are shown of ADSCs treated with TGF-β (TGF-β, 4d) or with bFGF (bFGF, 4d) for 4 days (left). SSFM containing bFGF was added to TGF-β-differentiated cells and TGF-β was added to bFGF-differentiated cells. Phase images were captured after 4 days (right). Images are representative of more than three independent experiments. Scale bar = 50 µm. (B) Lysates were prepared from cells grown under the six indicated conditions and were used for immunoblots with anti-α-SMA monoclonal antibody. GAPDH was used as the loading control. Blot is representative of three independent experiments. Legend indicates cell treatments in lanes 1–6. Unt., 4d = untreated for 4 days.

    Techniques Used: Western Blot

    Cytoskeletal responses to TGF-β and bFGF. Cell lysates prepared from ADSCs treated with TGFβ, bFGF or untreated (Unt) were subjected to SDS-PAGE and immunoblotted. (A) 4-day lysates were probed with anti-α-SMA monoclonal antibody or anti-GAPDH antibody as a loading control. Blot is representative of three independent experiments. (B) Lysates prepared at 1 hour of treatment were immunoblotted with anti-phospho-Smad2 (pSmad2) and with total Smad antibody. Blot is representative of three independent experiments. Dash represents 37 kD (A) or 50 kD (B). (C, D) After 4 days of treatment, cells were replated onto collagen-coated glass coverslips for two hours before fixation, permeabilization, and staining with anti-vinculin antibody (C, top), rhodamine-phalloidin (C, bottom), or anti-α-SMA monoclonal antibody (D). Insets in (D) show rhodamine-phalloidin staining of the same fields. All images are representative of three independent experiments. Scale bars = 50 µm.
    Figure Legend Snippet: Cytoskeletal responses to TGF-β and bFGF. Cell lysates prepared from ADSCs treated with TGFβ, bFGF or untreated (Unt) were subjected to SDS-PAGE and immunoblotted. (A) 4-day lysates were probed with anti-α-SMA monoclonal antibody or anti-GAPDH antibody as a loading control. Blot is representative of three independent experiments. (B) Lysates prepared at 1 hour of treatment were immunoblotted with anti-phospho-Smad2 (pSmad2) and with total Smad antibody. Blot is representative of three independent experiments. Dash represents 37 kD (A) or 50 kD (B). (C, D) After 4 days of treatment, cells were replated onto collagen-coated glass coverslips for two hours before fixation, permeabilization, and staining with anti-vinculin antibody (C, top), rhodamine-phalloidin (C, bottom), or anti-α-SMA monoclonal antibody (D). Insets in (D) show rhodamine-phalloidin staining of the same fields. All images are representative of three independent experiments. Scale bars = 50 µm.

    Techniques Used: SDS Page, Staining

    28) Product Images from "C. elegans Germ Cells Switch between Distinct Modes of Double-Strand Break Repair During Meiotic Prophase Progression"

    Article Title: C. elegans Germ Cells Switch between Distinct Modes of Double-Strand Break Repair During Meiotic Prophase Progression

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030191

    RAD-50 Dependence for Loading of RAD-51 at IR-Induced Breaks Extends from Meiotic Prophase Onset to the Mid-Pachytene/Late Pachytene Transition (A, B) Images show a portion of the germ lines of rad-50 mutant hermaphrodites fixed 1 h after exposure to 1 krad γ-irradiation, centered on the transition zone and extending from prior to the onset of meiotic prophase (premeiotic, left) through early pachytene (right). (A) HTP-3 staining is diffusely associated with chromatin in premeiotic nuclei, then becomes concentrated on chromosome axis structures at the beginning of the transition zone, first appearing as discontinuous stretches and then as extended continuous linear structures. IR-induced RAD-51 foci are abundant in nuclei with diffuse HTP-3 staining, whereas nuclei with concentrated meiotic HTP-3 signals have very few RAD-51 foci. Scale bar = 5 μm (B) SYP-1 immunostaining is either absent or detected as a single bright focus in premeiotic nuclei, and is extensively localized on chromosomes beginning in the transition zone. IR-induced RAD-51 foci are abundant in nuclei lacking SYP-1, whereas nuclei with abundant SYP-1 have very few RAD-51 foci. Scale bar = 5 μm. (C) An unirradiated wild-type gonad and an irradiated rad-50 gonad stained with DAPI, α-RAD-51, and a monoclonal antibody detecting the activated, diphosphorylated form of MAP kinase (MAPK). Left, zoomed out view of the gonads (distal tip through diplotene), showing the position of the peak of MAP kinase activation that occurs at the mid-pachytene to late pachytene transition. Right, zoomed in view of the region surrounding this peak of activated MAP kinase, showing the location of this peak relative to RAD-51 foci. In the unirradiated wild-type germ line, RAD-51 foci peak in mid-pachytene and diminish in numbers prior to the peak of activated MAPK. In the irradiated rad-50 germ line, background levels of bright SPO-11-independent spontaneous RAD-51 foci are seen in a subset of nuclei throughout the gonad, whereas IR-induced RAD-51 foci abruptly rise in abundance in late pachytene nuclei, after the peak of activated MAPK. Left; Scale bar = 20 μm. Right; Scale bar = 5 μm.
    Figure Legend Snippet: RAD-50 Dependence for Loading of RAD-51 at IR-Induced Breaks Extends from Meiotic Prophase Onset to the Mid-Pachytene/Late Pachytene Transition (A, B) Images show a portion of the germ lines of rad-50 mutant hermaphrodites fixed 1 h after exposure to 1 krad γ-irradiation, centered on the transition zone and extending from prior to the onset of meiotic prophase (premeiotic, left) through early pachytene (right). (A) HTP-3 staining is diffusely associated with chromatin in premeiotic nuclei, then becomes concentrated on chromosome axis structures at the beginning of the transition zone, first appearing as discontinuous stretches and then as extended continuous linear structures. IR-induced RAD-51 foci are abundant in nuclei with diffuse HTP-3 staining, whereas nuclei with concentrated meiotic HTP-3 signals have very few RAD-51 foci. Scale bar = 5 μm (B) SYP-1 immunostaining is either absent or detected as a single bright focus in premeiotic nuclei, and is extensively localized on chromosomes beginning in the transition zone. IR-induced RAD-51 foci are abundant in nuclei lacking SYP-1, whereas nuclei with abundant SYP-1 have very few RAD-51 foci. Scale bar = 5 μm. (C) An unirradiated wild-type gonad and an irradiated rad-50 gonad stained with DAPI, α-RAD-51, and a monoclonal antibody detecting the activated, diphosphorylated form of MAP kinase (MAPK). Left, zoomed out view of the gonads (distal tip through diplotene), showing the position of the peak of MAP kinase activation that occurs at the mid-pachytene to late pachytene transition. Right, zoomed in view of the region surrounding this peak of activated MAP kinase, showing the location of this peak relative to RAD-51 foci. In the unirradiated wild-type germ line, RAD-51 foci peak in mid-pachytene and diminish in numbers prior to the peak of activated MAPK. In the irradiated rad-50 germ line, background levels of bright SPO-11-independent spontaneous RAD-51 foci are seen in a subset of nuclei throughout the gonad, whereas IR-induced RAD-51 foci abruptly rise in abundance in late pachytene nuclei, after the peak of activated MAPK. Left; Scale bar = 20 μm. Right; Scale bar = 5 μm.

    Techniques Used: Mutagenesis, Irradiation, Staining, Immunostaining, Activation Assay

    29) Product Images from "Endothelial Function of von Hippel-Lindau Tumor Suppressor Gene: Control of Fibroblast Growth Factor Receptor Signaling"

    Article Title: Endothelial Function of von Hippel-Lindau Tumor Suppressor Gene: Control of Fibroblast Growth Factor Receptor Signaling

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-07-6003

    ERK1/2 pathway mediates cord formation by VHL knockdown HMVECs. A, top left , transfection of FGFR2 siRNA (siFGFR2) reduces expression of FGFR2 protein in both control and VHL knockdown HMVECs by ~ 3-fold. Bottom left and right , knockdown of FGFR2 (siFGFR2)
    Figure Legend Snippet: ERK1/2 pathway mediates cord formation by VHL knockdown HMVECs. A, top left , transfection of FGFR2 siRNA (siFGFR2) reduces expression of FGFR2 protein in both control and VHL knockdown HMVECs by ~ 3-fold. Bottom left and right , knockdown of FGFR2 (siFGFR2)

    Techniques Used: Transfection, Expressing

    30) Product Images from "An FGF-driven feed-forward circuit patterns the cardiopharyngeal mesoderm in space and time"

    Article Title: An FGF-driven feed-forward circuit patterns the cardiopharyngeal mesoderm in space and time

    Journal: eLife

    doi: 10.7554/eLife.29656

    Detailed patterns of MAPK activity during early cardiopharyngeal development. ( A ) MAPK activation during TVC induction. Close-up views of B7.5 lineage cells marked with Mesp > H2B::mCherry (nuclei) and Mesp > hCD4::mCherry (membranes) and immunostained for dpERK at indicated successive time points between 7 and 10hpf. DpERK staining was not detected in the founder cells at 7hpf, but increased sharply and specifically in the smaller trunk ventral cells (TVCs, open arrows) at 7.5hpf, but not in the larger anterior tail muscles (ATMs). DpERK staining persisted throughout TVC migration (see also B). ( B ) MAPK activation patterns during cardiopharyngeal fate diversification. DpERK staining was clearly detected in migrating TVCs (open arrows, 11 to 13hpf); in lateral large STVCs (open arrows, 14 to 15hpf), but not in the small median first heart precursors (FHPs, arrows, 14 to 15hpf); in the large lateral atrial siphon muscle founder cells (ASMFs, solid arrowheads, 16 to 17hpf), but neither in the FHPs (arrows), nor in the second heart precursors (SHPs, open arrowheads). ( C ) Treatment with the MEK inhibitor U0126 from 12 to 15.5hpf abolished dpERK staining in the lateral STVCs, compared to a control treatment with DMSO. Numbers of embryos showing the presented pattern out of the total numbers of embryos are shown.
    Figure Legend Snippet: Detailed patterns of MAPK activity during early cardiopharyngeal development. ( A ) MAPK activation during TVC induction. Close-up views of B7.5 lineage cells marked with Mesp > H2B::mCherry (nuclei) and Mesp > hCD4::mCherry (membranes) and immunostained for dpERK at indicated successive time points between 7 and 10hpf. DpERK staining was not detected in the founder cells at 7hpf, but increased sharply and specifically in the smaller trunk ventral cells (TVCs, open arrows) at 7.5hpf, but not in the larger anterior tail muscles (ATMs). DpERK staining persisted throughout TVC migration (see also B). ( B ) MAPK activation patterns during cardiopharyngeal fate diversification. DpERK staining was clearly detected in migrating TVCs (open arrows, 11 to 13hpf); in lateral large STVCs (open arrows, 14 to 15hpf), but not in the small median first heart precursors (FHPs, arrows, 14 to 15hpf); in the large lateral atrial siphon muscle founder cells (ASMFs, solid arrowheads, 16 to 17hpf), but neither in the FHPs (arrows), nor in the second heart precursors (SHPs, open arrowheads). ( C ) Treatment with the MEK inhibitor U0126 from 12 to 15.5hpf abolished dpERK staining in the lateral STVCs, compared to a control treatment with DMSO. Numbers of embryos showing the presented pattern out of the total numbers of embryos are shown.

    Techniques Used: Activity Assay, Activation Assay, Staining, Migration

    31) Product Images from "Neuroprotective Activity of Peripherally Administered Liver Growth Factor in a Rat Model of Parkinson's Disease"

    Article Title: Neuroprotective Activity of Peripherally Administered Liver Growth Factor in a Rat Model of Parkinson's Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067771

    Liver growth factor activated the MAPK/ERK1/2 signalling pathway and elicited the phosphorylation of CREB in the striatum of 6-OHDA-lesioned rats. A single injection of LGF promoted a transient increase in the phosphorylation of ERK1/2, which was observed 24 hours after the administration of the factor (A, B, lined bars). Moreover, 72 hours after LGF treatment, phospho-CREB levels were significantly raised (C, lined bars), as compared with control (C, dotted bar) and vehicle-treated rats (C, white bars). Lane 1: control; lane 2: lesioned striatum of vehicle rats; lane 3: lesioned striatum of 24-hour LGF-treated rats; lane 4: lesioned striatum of 48-hour LGF-treated rats; lane 5: lesioned striatum of 72-hour LGF-treated rats. Results are expressed as percentage of control (naïve striatum of IP-vehicle treated rats at 13 weeks post-lesion), and represent the mean ± SEM of n individual rats. One way ANOVA were performed in A (p = 0.0006; F 4, 28 = 6.791, n = 6–7), B (p = 0.0028; F 4, 29 = 5.178, n = 6–7) and C (p
    Figure Legend Snippet: Liver growth factor activated the MAPK/ERK1/2 signalling pathway and elicited the phosphorylation of CREB in the striatum of 6-OHDA-lesioned rats. A single injection of LGF promoted a transient increase in the phosphorylation of ERK1/2, which was observed 24 hours after the administration of the factor (A, B, lined bars). Moreover, 72 hours after LGF treatment, phospho-CREB levels were significantly raised (C, lined bars), as compared with control (C, dotted bar) and vehicle-treated rats (C, white bars). Lane 1: control; lane 2: lesioned striatum of vehicle rats; lane 3: lesioned striatum of 24-hour LGF-treated rats; lane 4: lesioned striatum of 48-hour LGF-treated rats; lane 5: lesioned striatum of 72-hour LGF-treated rats. Results are expressed as percentage of control (naïve striatum of IP-vehicle treated rats at 13 weeks post-lesion), and represent the mean ± SEM of n individual rats. One way ANOVA were performed in A (p = 0.0006; F 4, 28 = 6.791, n = 6–7), B (p = 0.0028; F 4, 29 = 5.178, n = 6–7) and C (p

    Techniques Used: Injection

    32) Product Images from "Anti-Inflammatory and Anti-Fibrotic Profile of Fish Oil Emulsions Used in Parenteral Nutrition-Associated Liver Disease"

    Article Title: Anti-Inflammatory and Anti-Fibrotic Profile of Fish Oil Emulsions Used in Parenteral Nutrition-Associated Liver Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115404

    Omegaven® inhibits epithelial to mesenchymal transition induced by TGFβ1. Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by TGFβ1 5 ng/mL stimulation for additional 72 hours (A and B) or 25 min (C). (A) Expression of mRNA of ZO-1 and (B) E-cadherin . (C) Phosphorylation of Samd3, ERK1/2 and Akt and nuclear expression of β-catenin. Representative western blot are showed and quantified in graphic bars. Results are expressed as means ± SEM of six independent experiments. * p
    Figure Legend Snippet: Omegaven® inhibits epithelial to mesenchymal transition induced by TGFβ1. Human liver epithelial cell line THLE-3 was incubated in presence or absence of lipid emulsions Omegaven® 10%, Lipofundin MCT/LCT® 20%, ClinOleic® 20% or SMOFlipid® 20% at different dilutions, for 30 min followed by TGFβ1 5 ng/mL stimulation for additional 72 hours (A and B) or 25 min (C). (A) Expression of mRNA of ZO-1 and (B) E-cadherin . (C) Phosphorylation of Samd3, ERK1/2 and Akt and nuclear expression of β-catenin. Representative western blot are showed and quantified in graphic bars. Results are expressed as means ± SEM of six independent experiments. * p

    Techniques Used: Incubation, Expressing, Western Blot

    33) Product Images from "Distinct Cell Cycle Timing Requirements for Extracellular Signal-Regulated Kinase and Phosphoinositide 3-Kinase Signaling Pathways in Somatic Cell Mitosis"

    Article Title: Distinct Cell Cycle Timing Requirements for Extracellular Signal-Regulated Kinase and Phosphoinositide 3-Kinase Signaling Pathways in Somatic Cell Mitosis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.20.7226-7241.2002

    Activation of ERK during S/G 2 /M. Cells were synchronized by thymidine arrest, released, and harvested at the indicated times by scraping, and lysates (20 μg) were immunoblotted with an anti-ppERK or anti-ERK2 antibody. In parallel experiments, cells were analyzed by flow cytometry. (A) ERK1/2 is diphosphorylated in NIH 3T3 cells within 30 min after release and inhibited by treating cells at 0 h with 15 μM U-0126 or 75 μM PD-98059. Controls in these and other experiments were treated with an equal volume of DMSO carrier. (B) ERK1/2 is diphosphorylated in HeLa cells within 3 h after release, decreases as cells progress through G 2 /M (6 to 8.5 h), and increases again as cells enter G 1 . ERK phosphorylation was inhibited strongly by 25 μM U-0126 and weakly by 100 μM PD-98059.
    Figure Legend Snippet: Activation of ERK during S/G 2 /M. Cells were synchronized by thymidine arrest, released, and harvested at the indicated times by scraping, and lysates (20 μg) were immunoblotted with an anti-ppERK or anti-ERK2 antibody. In parallel experiments, cells were analyzed by flow cytometry. (A) ERK1/2 is diphosphorylated in NIH 3T3 cells within 30 min after release and inhibited by treating cells at 0 h with 15 μM U-0126 or 75 μM PD-98059. Controls in these and other experiments were treated with an equal volume of DMSO carrier. (B) ERK1/2 is diphosphorylated in HeLa cells within 3 h after release, decreases as cells progress through G 2 /M (6 to 8.5 h), and increases again as cells enter G 1 . ERK phosphorylation was inhibited strongly by 25 μM U-0126 and weakly by 100 μM PD-98059.

    Techniques Used: Activation Assay, Flow Cytometry, Cytometry

    Related Articles

    Electrophoresis:

    Article Title: Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans
    Article Snippet: .. Immunoblotting of MPK-1 after electrophoresis on 12% SDS–polyacrylamide gels used either polyclonal rabbit pan-ERK antibody (Santa Cruz sc-153) at 1:500 or monoclonal anti-pTpY-ERK (Sigma M-8159) at 1:2000 with detection by peroxidase-labeled donkey secondary antibodies and TMB substrate (KPL Laboratories). .. Animals were stained for β-galactosidase activity with 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-gal) as described ( ) for 1–2 h at room temperature, governed by visual examination of stained control animals (wild type or mutants at permissive temperature) included with every experiment.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Unc-51 controls active zone density and protein composition by downregulating ERK signaling
    Article Snippet: .. Mouse anti-diphospho ERK (Sigma, St. Louis, MO, CatM8159) and mouse anti-phospho p38 (cell Signaling Technology, Inc., Boston, MA) were used at 1:1000. ..

    SDS Page:

    Article Title: Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses
    Article Snippet: .. Antibodies and Reagents Mouse anti-LAMP1 monoclonal antibody (555798) was purchased from BD Biosciences; mouse anti-diphosphorylated ERK1/2 monoclonal antibody (M8159) was from Sigma-Aldrich; rabbit anti-ERK1/2 polyclonal antibody (V114A) was from Promega; mouse anti-β-actin monoclonal antibody (G043) was from Abm; mouse anti-γ-tubulin antibody (T6557) was from Sigma-Aldrich; goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) (sc-2031) and goat anti-rabbit IgG-HRP polyclonal antibody (sc-3837) were from Santa Cruz Biotechnology; goat anti-mouse IgG-TRITC antibody (T5393) was from Sigma-Aldrich; BSA (A7906) was from Sigma-Aldrich; SDS-PAGE reagents were from Bio-Rad; fetal bovine serum (FBS) was from Gibco; LysoTracker (L7528) was from Thermo Fisher Scientific; fibronectin (F2006) was from Sigma-Aldrich; FGF2 was from PeproTech; and Alcian blue dye (74240) was from EuroDiagnostica. .. The recombinant NK1 fragment of HGF was produced using yeast Pichia pastoris expression system and purified with heparin affinity chromatography as previously described.

    Article Title: Targeting Heparan Sulfate Proteoglycans as a Novel Therapeutic Strategy for Mucopolysaccharidoses
    Article Snippet: .. Mouse anti-LAMP1 monoclonal antibody (555798) was purchased from BD Biosciences; mouse anti-diphosphorylated ERK1/2 monoclonal antibody (M8159) was from Sigma-Aldrich; rabbit anti-ERK1/2 polyclonal antibody (V114A) was from Promega; mouse anti-β-actin monoclonal antibody (G043) was from Abm; mouse anti-γ-tubulin antibody (T6557) was from Sigma-Aldrich; goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) (sc-2031) and goat anti-rabbit IgG-HRP polyclonal antibody (sc-3837) were from Santa Cruz Biotechnology; goat anti-mouse IgG-TRITC antibody (T5393) was from Sigma-Aldrich; BSA (A7906) was from Sigma-Aldrich; SDS-PAGE reagents were from Bio-Rad; fetal bovine serum (FBS) was from Gibco; LysoTracker (L7528) was from Thermo Fisher Scientific; fibronectin (F2006) was from Sigma-Aldrich; FGF2 was from PeproTech; and Alcian blue dye (74240) was from EuroDiagnostica. .. The recombinant NK1 fragment of HGF was produced using yeast Pichia pastoris expression system and purified with heparin affinity chromatography as previously described.

    Molecular Weight:

    Article Title: Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6
    Article Snippet: .. Membranes were probed with antibodies targeting total and phosphorylated forms of ERK1/2 (anti-ERK1/2 [AB17942; [Abcam] and anti-MAPK activated diphosphorylated ERK1/2 [M8159; Sigma-Aldrich]), p38 (MAPK p38/MAPK14 [AP03041SU-N; Acris antibodies, 2B Scientific] and MAPK p38/MAPK14pThr180/pTyr182 [AP05898PU-N, Acris antibodies, 2B Scientific]), and NFκB (NFκB p65 [4764S; New England Biolabs] and phospho NFκB p65(Ser536) [3033L; New England Biolabs]), with protein loading confirmed using antibodies against α-tubulin (bovine α-tubulin, A11126; Invitrogen) or β-actin (bovine anti-β actin, ab8226; Abcam); the antibodies are further detailed in Supplemental Table 1 and were selected on the basis of recognition of immunoreactive proteins of appropriate molecular weight and previous publications ( , , ). .. Membranes were incubated with primary antibodies diluted in 5% (wt/vol) BSA in TBS/T for 2 hours with gentle agitation.

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    Millipore erk1 2
    (A) IKKγ expression rescues LPS- and IL-1β- but not PMA-induced NF-κB activation of 1.3E2 cells. 70Z/3 cells, three 1.3E2 IKKγ-expressing clones, and 1.3E2 cells were treated with PMA, LPS, or IL-1β for 30 min and NF-κB DNA-binding activity was determined by EMSA. IKKγ protein was determined by Western blotting (lower panel). (B and C) Activation of IKK and JNK1 in response to PMA is defective in 1.3E2 and 1.3E2 IKKγ cells. (B) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), and extracts were analyzed for IKKα protein expression. IKK activity was determined in an in vitro kinase reaction after IKK immunoprecipitation with an anti-IKKα antibody. GstIκBα1–53 was used as a substrate. (C) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), LPS (20 min), or UV light (20 min) and cellular extracts were analyzed for JNK1 expression (upper panel). The hyperphosphorylated 46-kDa JNK1 isoform is indicated by a star. JNK1 kinase activity was determined in an in vitro kinase reaction after immunoprecipitation of JNK1. Kinase activity was determined with recombinant GstJun1–79 as substrate. (D) Activation of <t>ERK1/2</t> is not defective in 1.3E2 cells. Cells were stimulated with PMA for 10 min and cellular extracts were analyzed by Western blotting using JNK1, phospho-ERK1/2, or ERK1/2 antibodies (as indicated). Migration of the hyperphosphorylated 46-kDa JNK1 is indicated by a star.
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    (A) IKKγ expression rescues LPS- and IL-1β- but not PMA-induced NF-κB activation of 1.3E2 cells. 70Z/3 cells, three 1.3E2 IKKγ-expressing clones, and 1.3E2 cells were treated with PMA, LPS, or IL-1β for 30 min and NF-κB DNA-binding activity was determined by EMSA. IKKγ protein was determined by Western blotting (lower panel). (B and C) Activation of IKK and JNK1 in response to PMA is defective in 1.3E2 and 1.3E2 IKKγ cells. (B) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), and extracts were analyzed for IKKα protein expression. IKK activity was determined in an in vitro kinase reaction after IKK immunoprecipitation with an anti-IKKα antibody. GstIκBα1–53 was used as a substrate. (C) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), LPS (20 min), or UV light (20 min) and cellular extracts were analyzed for JNK1 expression (upper panel). The hyperphosphorylated 46-kDa JNK1 isoform is indicated by a star. JNK1 kinase activity was determined in an in vitro kinase reaction after immunoprecipitation of JNK1. Kinase activity was determined with recombinant GstJun1–79 as substrate. (D) Activation of ERK1/2 is not defective in 1.3E2 cells. Cells were stimulated with PMA for 10 min and cellular extracts were analyzed by Western blotting using JNK1, phospho-ERK1/2, or ERK1/2 antibodies (as indicated). Migration of the hyperphosphorylated 46-kDa JNK1 is indicated by a star.

    Journal: Molecular and Cellular Biology

    Article Title: B-Cell Receptor- and Phorbol Ester-Induced NF-?B and c-Jun N-Terminal Kinase Activation in B Cells Requires Novel Protein Kinase C's

    doi: 10.1128/MCB.21.19.6640-6650.2001

    Figure Lengend Snippet: (A) IKKγ expression rescues LPS- and IL-1β- but not PMA-induced NF-κB activation of 1.3E2 cells. 70Z/3 cells, three 1.3E2 IKKγ-expressing clones, and 1.3E2 cells were treated with PMA, LPS, or IL-1β for 30 min and NF-κB DNA-binding activity was determined by EMSA. IKKγ protein was determined by Western blotting (lower panel). (B and C) Activation of IKK and JNK1 in response to PMA is defective in 1.3E2 and 1.3E2 IKKγ cells. (B) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), and extracts were analyzed for IKKα protein expression. IKK activity was determined in an in vitro kinase reaction after IKK immunoprecipitation with an anti-IKKα antibody. GstIκBα1–53 was used as a substrate. (C) 70Z/3, 1.3E2, and 1.3E2 IKKγ2 cells were stimulated with PMA (10 min), LPS (20 min), or UV light (20 min) and cellular extracts were analyzed for JNK1 expression (upper panel). The hyperphosphorylated 46-kDa JNK1 isoform is indicated by a star. JNK1 kinase activity was determined in an in vitro kinase reaction after immunoprecipitation of JNK1. Kinase activity was determined with recombinant GstJun1–79 as substrate. (D) Activation of ERK1/2 is not defective in 1.3E2 cells. Cells were stimulated with PMA for 10 min and cellular extracts were analyzed by Western blotting using JNK1, phospho-ERK1/2, or ERK1/2 antibodies (as indicated). Migration of the hyperphosphorylated 46-kDa JNK1 is indicated by a star.

    Article Snippet: Further antibodies used were monoclonal JNK1 (Pharmingen), phospho-SAPK/JNK (Cell Signaling), ERK1/2 (Calbiochem), and phospho-ERK1/2 (Biomol).

    Techniques: Expressing, Activation Assay, Clone Assay, Binding Assay, Activity Assay, Western Blot, In Vitro, Immunoprecipitation, Recombinant, Migration