anti total erk  (Cell Signaling Technology Inc)

 
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    Name:
    p44 42 MAPK Erk1 2 Antibody
    Description:
    Mitogen activated protein kinases MAPKs are a widely conserved family of serine threonine protein kinases involved in many cellular programs such as cell proliferation differentiation motility and death The p44 42 MAPK Erk1 2 signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens growth factors and cytokines 1 3 and research investigators consider it an important target in the diagnosis and treatment of cancer 4 Upon stimulation a sequential three part protein kinase cascade is initiated consisting of a MAP kinase kinase kinase MAPKKK or MAP3K a MAP kinase kinase MAPKK or MAP2K and a MAP kinase MAPK Multiple p44 42 MAP3Ks have been identified including members of the Raf family as well as Mos and Tpl2 COT MEK1 and MEK2 are the primary MAPKKs in this pathway 5 6 MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202 Tyr204 and Thr185 Tyr187 respectively Several downstream targets of p44 42 have been identified including p90RSK 7 and the transcription factor Elk 1 8 9 p44 42 are negatively regulated by a family of dual specificity Thr Tyr MAPK phosphatases known as DUSPs or MKPs 10 along with MEK inhibitors such as U0126 and PD98059
    Catalog Number:
    9102
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunohistochemistry
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a sequence in the C-terminus of rat p44 MAP Kinase. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Hamster Monkey Mink Zebrafish Bovine Pig S cerevisiae
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    Structured Review

    Cell Signaling Technology Inc anti total erk
    The <t>miR-630/YAP1/ERK</t> feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of <t>p-AKT,</t> total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P
    Mitogen activated protein kinases MAPKs are a widely conserved family of serine threonine protein kinases involved in many cellular programs such as cell proliferation differentiation motility and death The p44 42 MAPK Erk1 2 signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens growth factors and cytokines 1 3 and research investigators consider it an important target in the diagnosis and treatment of cancer 4 Upon stimulation a sequential three part protein kinase cascade is initiated consisting of a MAP kinase kinase kinase MAPKKK or MAP3K a MAP kinase kinase MAPKK or MAP2K and a MAP kinase MAPK Multiple p44 42 MAP3Ks have been identified including members of the Raf family as well as Mos and Tpl2 COT MEK1 and MEK2 are the primary MAPKKs in this pathway 5 6 MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202 Tyr204 and Thr185 Tyr187 respectively Several downstream targets of p44 42 have been identified including p90RSK 7 and the transcription factor Elk 1 8 9 p44 42 are negatively regulated by a family of dual specificity Thr Tyr MAPK phosphatases known as DUSPs or MKPs 10 along with MEK inhibitors such as U0126 and PD98059
    https://www.bioz.com/result/anti total erk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    anti total erk - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop"

    Article Title: A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop

    Journal: Theranostics

    doi: 10.7150/thno.22048

    The miR-630/YAP1/ERK feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of p-AKT, total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P
    Figure Legend Snippet: The miR-630/YAP1/ERK feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of p-AKT, total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Over Expression, Staining, Flow Cytometry, Cytometry, Western Blot

    2) Product Images from "Activation of a novel estrogen receptor, GPER, is cardioprotective in male and female rats"

    Article Title: Activation of a novel estrogen receptor, GPER, is cardioprotective in male and female rats

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00283.2009

    Stimulation of GPER resulted in activation of downstream signaling molecules. Ratios of phosphorylated (p) Akt to total Akt and phosphorylated ERK to total ERK levels were analyzed. Top : representative immunoblots of phosphorylated Akt, total Akt, phosphorylated
    Figure Legend Snippet: Stimulation of GPER resulted in activation of downstream signaling molecules. Ratios of phosphorylated (p) Akt to total Akt and phosphorylated ERK to total ERK levels were analyzed. Top : representative immunoblots of phosphorylated Akt, total Akt, phosphorylated

    Techniques Used: Activation Assay, Western Blot

    3) Product Images from "Reprofiling using a zebrafish melanoma model reveals drugs cooperating with targeted therapeutics"

    Article Title: Reprofiling using a zebrafish melanoma model reveals drugs cooperating with targeted therapeutics

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9613

    Molecular characterization of the V12RAS model a–d. Transverse sections of V12RAS larvae. (a) H E staining. Scale bar = 0.2 mm. (b–d) IHC on bleached sections. (b) Non-specific primary control, (c) phospho-ERK and (d) phospho-Akt. Positive stain (brown) denoted by asterisks. e. Results of immunoblotting protein extract (30 μg) from 30 pooled 5-dpf embryos exposed for 4 h to the specified drug at the indicated concentrations. Representative immunoblots are depicted with densitometric quantification shown immediately below (mean p-protein/total protein ± SEM for three independent experiments). *P
    Figure Legend Snippet: Molecular characterization of the V12RAS model a–d. Transverse sections of V12RAS larvae. (a) H E staining. Scale bar = 0.2 mm. (b–d) IHC on bleached sections. (b) Non-specific primary control, (c) phospho-ERK and (d) phospho-Akt. Positive stain (brown) denoted by asterisks. e. Results of immunoblotting protein extract (30 μg) from 30 pooled 5-dpf embryos exposed for 4 h to the specified drug at the indicated concentrations. Representative immunoblots are depicted with densitometric quantification shown immediately below (mean p-protein/total protein ± SEM for three independent experiments). *P

    Techniques Used: Staining, Immunohistochemistry, Western Blot

    4) Product Images from "Dioscin overcome TKI resistance in EGFR-mutated lung adenocarcinoma cells via down-regulation of tyrosine phosphatase SHP2 expression"

    Article Title: Dioscin overcome TKI resistance in EGFR-mutated lung adenocarcinoma cells via down-regulation of tyrosine phosphatase SHP2 expression

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.22209

    Dioscin overcomes TKI resistance via simultaneous inhibition of the MEK/ERK and PI3K/AKT signaling pathways due to decreased SHP2 expression and its interaction with GAB1. (a) The effect of dioscin on AKT and ERK signaling. Five hours after dioscin (-5 μM) treatment, the cell lysates were harvested and analyzed for signaling alteration by weocistern blotting with the indicated antibodies. (b) The effect of dioscin on SHP2 transcriptional level. Five hours after dosing cells with dioscin (0-5 μM), the cell lysates were prepared and analyzed for mRNA and promoter activity by real-time PCR and luciferase assays. The relative luciferase activity and mRNA expression was shown as fold-activation relative to the cells with vehicle treatment. (c) The effect of SHP2 knockdown on changes in cell viability and apoptosis induced by dioscin treatment. H1975, CL97, H1650, and PC9GR cells transfected with SHP2 shRNA were treated with 5 μM dioscin for 48 h. The cell viability was measured by MTT assays. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. (d) The effect of SHP2 overexpression or/and GAB1 knockdown on the apoptosis induced by dioscin treatment. H1975 and CL97 cells transfected with SHP2 overexpression plasmid or/and GAB1 shRNA were treated with 5 μM dioscin for 48 h. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. All data were collected from three independent experiments. The mean value and standard deviation are indicated as the column with error bars . The significant differences (*P
    Figure Legend Snippet: Dioscin overcomes TKI resistance via simultaneous inhibition of the MEK/ERK and PI3K/AKT signaling pathways due to decreased SHP2 expression and its interaction with GAB1. (a) The effect of dioscin on AKT and ERK signaling. Five hours after dioscin (-5 μM) treatment, the cell lysates were harvested and analyzed for signaling alteration by weocistern blotting with the indicated antibodies. (b) The effect of dioscin on SHP2 transcriptional level. Five hours after dosing cells with dioscin (0-5 μM), the cell lysates were prepared and analyzed for mRNA and promoter activity by real-time PCR and luciferase assays. The relative luciferase activity and mRNA expression was shown as fold-activation relative to the cells with vehicle treatment. (c) The effect of SHP2 knockdown on changes in cell viability and apoptosis induced by dioscin treatment. H1975, CL97, H1650, and PC9GR cells transfected with SHP2 shRNA were treated with 5 μM dioscin for 48 h. The cell viability was measured by MTT assays. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. (d) The effect of SHP2 overexpression or/and GAB1 knockdown on the apoptosis induced by dioscin treatment. H1975 and CL97 cells transfected with SHP2 overexpression plasmid or/and GAB1 shRNA were treated with 5 μM dioscin for 48 h. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. All data were collected from three independent experiments. The mean value and standard deviation are indicated as the column with error bars . The significant differences (*P

    Techniques Used: Inhibition, Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Activation Assay, Transfection, shRNA, MTT Assay, Staining, Flow Cytometry, Cytometry, Over Expression, Plasmid Preparation, Standard Deviation

    5) Product Images from "Effect of Urban Particulate Matter on Vocal Fold Fibrosis through the MAPK/NF-κB Signaling Pathway"

    Article Title: Effect of Urban Particulate Matter on Vocal Fold Fibrosis through the MAPK/NF-κB Signaling Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21186643

    PM induces activity in the NF-κB signaling pathway through the MAPK pathway. ( A ) The expression levels of the MAPK family members ERK, JNK, and P38 were evaluated by Western blot. The levels of p-ERK, p-JNK, and p-P38 expression increased according to the PM concentration. Cells were treated with various concentrations of PM (0–50 μg/mL) for 12 h. ( B ) Increased expression of the protein associated with the activation of the MAPK signaling pathway was reduced by specific inhibitors (ERK inhibitor: PD98059; JNK inhibitor: SP600125; P38 inhibitor: SB203580). The increased expression of p-P65 after the PM treatment was also reduced by the inhibitors. Cells were treated with PM (20 μg/mL) for 12 h.
    Figure Legend Snippet: PM induces activity in the NF-κB signaling pathway through the MAPK pathway. ( A ) The expression levels of the MAPK family members ERK, JNK, and P38 were evaluated by Western blot. The levels of p-ERK, p-JNK, and p-P38 expression increased according to the PM concentration. Cells were treated with various concentrations of PM (0–50 μg/mL) for 12 h. ( B ) Increased expression of the protein associated with the activation of the MAPK signaling pathway was reduced by specific inhibitors (ERK inhibitor: PD98059; JNK inhibitor: SP600125; P38 inhibitor: SB203580). The increased expression of p-P65 after the PM treatment was also reduced by the inhibitors. Cells were treated with PM (20 μg/mL) for 12 h.

    Techniques Used: Activity Assay, Expressing, Western Blot, Concentration Assay, Activation Assay

    6) Product Images from "Dioscin overcome TKI resistance in EGFR-mutated lung adenocarcinoma cells via down-regulation of tyrosine phosphatase SHP2 expression"

    Article Title: Dioscin overcome TKI resistance in EGFR-mutated lung adenocarcinoma cells via down-regulation of tyrosine phosphatase SHP2 expression

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.22209

    Dioscin overcomes TKI resistance via simultaneous inhibition of the MEK/ERK and PI3K/AKT signaling pathways due to decreased SHP2 expression and its interaction with GAB1. (a) The effect of dioscin on AKT and ERK signaling. Five hours after dioscin (-5 μM) treatment, the cell lysates were harvested and analyzed for signaling alteration by weocistern blotting with the indicated antibodies. (b) The effect of dioscin on SHP2 transcriptional level. Five hours after dosing cells with dioscin (0-5 μM), the cell lysates were prepared and analyzed for mRNA and promoter activity by real-time PCR and luciferase assays. The relative luciferase activity and mRNA expression was shown as fold-activation relative to the cells with vehicle treatment. (c) The effect of SHP2 knockdown on changes in cell viability and apoptosis induced by dioscin treatment. H1975, CL97, H1650, and PC9GR cells transfected with SHP2 shRNA were treated with 5 μM dioscin for 48 h. The cell viability was measured by MTT assays. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. (d) The effect of SHP2 overexpression or/and GAB1 knockdown on the apoptosis induced by dioscin treatment. H1975 and CL97 cells transfected with SHP2 overexpression plasmid or/and GAB1 shRNA were treated with 5 μM dioscin for 48 h. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. All data were collected from three independent experiments. The mean value and standard deviation are indicated as the column with error bars . The significant differences (*P
    Figure Legend Snippet: Dioscin overcomes TKI resistance via simultaneous inhibition of the MEK/ERK and PI3K/AKT signaling pathways due to decreased SHP2 expression and its interaction with GAB1. (a) The effect of dioscin on AKT and ERK signaling. Five hours after dioscin (-5 μM) treatment, the cell lysates were harvested and analyzed for signaling alteration by weocistern blotting with the indicated antibodies. (b) The effect of dioscin on SHP2 transcriptional level. Five hours after dosing cells with dioscin (0-5 μM), the cell lysates were prepared and analyzed for mRNA and promoter activity by real-time PCR and luciferase assays. The relative luciferase activity and mRNA expression was shown as fold-activation relative to the cells with vehicle treatment. (c) The effect of SHP2 knockdown on changes in cell viability and apoptosis induced by dioscin treatment. H1975, CL97, H1650, and PC9GR cells transfected with SHP2 shRNA were treated with 5 μM dioscin for 48 h. The cell viability was measured by MTT assays. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. (d) The effect of SHP2 overexpression or/and GAB1 knockdown on the apoptosis induced by dioscin treatment. H1975 and CL97 cells transfected with SHP2 overexpression plasmid or/and GAB1 shRNA were treated with 5 μM dioscin for 48 h. Cell apoptosis was measured by annexinV-PI staining assays and flow cytometry. All data were collected from three independent experiments. The mean value and standard deviation are indicated as the column with error bars . The significant differences (*P

    Techniques Used: Inhibition, Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Activation Assay, Transfection, shRNA, MTT Assay, Staining, Flow Cytometry, Cytometry, Over Expression, Plasmid Preparation, Standard Deviation

    7) Product Images from "DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori"

    Article Title: DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms16036266

    Insulin-activated DNA synthesis of silk gland cells is dependent on PI3K/Akt and TOR signaling, but not ERK signaling. The silk glands of day 1 fourth instar larvae were preincubated with LY294002 (48.8 μM), rapamycin (5.5 μM) or U0126 (21 μM) for 45 min and then incubated with medium containing insulin (1.74 μM) with or without LY294002 (48.8 μM) ( A ), rapamycin (5.5 μM) ( B ) or U0126 (21 μM) ( C ) for 1 h. The silk glands were labeled with BrdU and then analyzed. After preincubation for 45 min, the silk glands were incubated with medium containing 1.74 μM insulin with or without LY294002 ( D ), rapamycin ( E ) or U0126 ( F ) for 15 min. Silk gland lysates were prepared and subjected to an immunoblot analysis with their corresponding antibodies. The results shown are representative of three independent experiments. Con: control; Ins: insulin; LY: LY294002; R: rapamycin; U: U0126. * p
    Figure Legend Snippet: Insulin-activated DNA synthesis of silk gland cells is dependent on PI3K/Akt and TOR signaling, but not ERK signaling. The silk glands of day 1 fourth instar larvae were preincubated with LY294002 (48.8 μM), rapamycin (5.5 μM) or U0126 (21 μM) for 45 min and then incubated with medium containing insulin (1.74 μM) with or without LY294002 (48.8 μM) ( A ), rapamycin (5.5 μM) ( B ) or U0126 (21 μM) ( C ) for 1 h. The silk glands were labeled with BrdU and then analyzed. After preincubation for 45 min, the silk glands were incubated with medium containing 1.74 μM insulin with or without LY294002 ( D ), rapamycin ( E ) or U0126 ( F ) for 15 min. Silk gland lysates were prepared and subjected to an immunoblot analysis with their corresponding antibodies. The results shown are representative of three independent experiments. Con: control; Ins: insulin; LY: LY294002; R: rapamycin; U: U0126. * p

    Techniques Used: DNA Synthesis, Incubation, Labeling

    A predicted network that links the growth factor signaling pathways with silk gland cell DNA synthesis in the silkworm. Insulin activated silk gland DNA synthesis via PI3K/AKT/TOR signaling. MAPK/ERK signaling may be activated by other growth factor(s). Solid lines indicate demonstrable or highly likely relations; dashed lines indicate hypothetical interactions.
    Figure Legend Snippet: A predicted network that links the growth factor signaling pathways with silk gland cell DNA synthesis in the silkworm. Insulin activated silk gland DNA synthesis via PI3K/AKT/TOR signaling. MAPK/ERK signaling may be activated by other growth factor(s). Solid lines indicate demonstrable or highly likely relations; dashed lines indicate hypothetical interactions.

    Techniques Used: DNA Synthesis

    8) Product Images from "Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis"

    Article Title: Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00032.2014

    Active p-ERK levels are decreased in Sec10-overexpressing cells following addition of gefitinib. Sec10-overexpressing and control MDCK cells were grown on Transwell filters for 7 days. Under basal conditions, specific inhibition of EGFR kinase activity
    Figure Legend Snippet: Active p-ERK levels are decreased in Sec10-overexpressing cells following addition of gefitinib. Sec10-overexpressing and control MDCK cells were grown on Transwell filters for 7 days. Under basal conditions, specific inhibition of EGFR kinase activity

    Techniques Used: Inhibition, Activity Assay

    9) Product Images from "Inhibition of lung carcinogenesis and critical cancer-related signaling pathways by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-l-cysteine, indole-3-carbinol and myo-inositol, alone and in combination"

    Article Title: Inhibition of lung carcinogenesis and critical cancer-related signaling pathways by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-l-cysteine, indole-3-carbinol and myo-inositol, alone and in combination

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgq139

    Modulation by PEITC-NAC, DIM and MI, alone and in combination, of p-Akt, p-ERK and p-NF-κB expression in mouse lung tissues ( A ) HBEC ( B ) and A549 cells ( C ). Lung tissue lysates were prepared from aliquots of normal lungs (lane 1) or lung tumors from mice treated with NNK plus BaP (lane 2) or NNK plus BaP plus chemopreventive agents, alone or in combination. (lanes 3–9). Samples from HBEC were prepared from cells pretreated with DMSO (lane 1) or CSC (lane 2) for 12 weeks or cells pretreated with CSC for 12 weeks and exposed to PEITC-NAC (1 μM), DIM (2 μM) and MI (300 μM), alone or in combination, for 2 weeks (lanes 3–9). Lysates from A549 cells were prepared after treatment with DMSO (lane 1) or PEITC-NAC (5 μM), DIM (10 μM) and MI (300 μM), alone or in combination, for 1 week (lanes 2–8). Samples (40 μg protein) were resolved on a 4–12% sodium dodecyl sulfate–polyacrylamide electrophoresis followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. Relative fold of expression of the proteins was determined by densitometry analysis. * P
    Figure Legend Snippet: Modulation by PEITC-NAC, DIM and MI, alone and in combination, of p-Akt, p-ERK and p-NF-κB expression in mouse lung tissues ( A ) HBEC ( B ) and A549 cells ( C ). Lung tissue lysates were prepared from aliquots of normal lungs (lane 1) or lung tumors from mice treated with NNK plus BaP (lane 2) or NNK plus BaP plus chemopreventive agents, alone or in combination. (lanes 3–9). Samples from HBEC were prepared from cells pretreated with DMSO (lane 1) or CSC (lane 2) for 12 weeks or cells pretreated with CSC for 12 weeks and exposed to PEITC-NAC (1 μM), DIM (2 μM) and MI (300 μM), alone or in combination, for 2 weeks (lanes 3–9). Lysates from A549 cells were prepared after treatment with DMSO (lane 1) or PEITC-NAC (5 μM), DIM (10 μM) and MI (300 μM), alone or in combination, for 1 week (lanes 2–8). Samples (40 μg protein) were resolved on a 4–12% sodium dodecyl sulfate–polyacrylamide electrophoresis followed by immunoblot analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. Relative fold of expression of the proteins was determined by densitometry analysis. * P

    Techniques Used: Expressing, Mouse Assay, Electrophoresis, Stripping Membranes

    10) Product Images from "A critical role for DAP10 and DAP12 in CD8+ T cell–mediated tissue damage in large granular lymphocyte leukemia"

    Article Title: A critical role for DAP10 and DAP12 in CD8+ T cell–mediated tissue damage in large granular lymphocyte leukemia

    Journal: Blood

    doi: 10.1182/blood-2008-07-168245

    CD8 + T cells from LGLL patients maintain constitutively activated ERK1/2 and PI3K . CD8 + T-cell lysates were prepared from LGLL patients and healthy donors. The activity of ERK and PI3K in these cells was assessed by Western blot analyses using antibodies specific for (A) phosphorylated ERK1/2 (pERK) and (C) phosphorylated AKT. Membranes were stripped and reprobed with antibodies that detect (B) pan-ERK and (D) pan-AKT to demonstrate total protein levels.
    Figure Legend Snippet: CD8 + T cells from LGLL patients maintain constitutively activated ERK1/2 and PI3K . CD8 + T-cell lysates were prepared from LGLL patients and healthy donors. The activity of ERK and PI3K in these cells was assessed by Western blot analyses using antibodies specific for (A) phosphorylated ERK1/2 (pERK) and (C) phosphorylated AKT. Membranes were stripped and reprobed with antibodies that detect (B) pan-ERK and (D) pan-AKT to demonstrate total protein levels.

    Techniques Used: Activity Assay, Western Blot

    11) Product Images from "Chronic Corticosterone Exposure Persistently Elevates the Expression of Memory-Related Genes in the Lateral Amygdala and Enhances the Consolidation of a Pavlovian Fear Memory"

    Article Title: Chronic Corticosterone Exposure Persistently Elevates the Expression of Memory-Related Genes in the Lateral Amygdala and Enhances the Consolidation of a Pavlovian Fear Memory

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091530

    Chronic exposure to CORT persistently enhances the expression of memory-related IEGs and synaptically-localized proteins in the LA. ( A ) Schematic of the behavioral protocol. Rats received either Water or CORT in their drinking water (50 μg/ml) for 2 weeks. Half the rats were sacrificed at the end of CORT exposure period. The other half was titrated off the CORT (25 μg/ml, 12.5 μg/ml) and given a 2 week period of exposure to water alone (‘wash-out’) prior to being sacrificed. ( B ) Mean (±SEM) immunoreactivity for GluR1, synaptophysin and BDNF (Water: n = 9; CORT: n = 9) from area CA3 in rats sacrificed on the last day of CORT exposure. ( C ) Mean (±SEM) immunoreactivity for GluR1 (Water: n = 9; CORT: n = 9), synaptophysin (Water: n = 9; CORT: n = 9), Arc/Arg3.1 (Water: n = 9; CORT: n = 9), Egr-1 (Water: n = 9; CORT: n = 9), BDNF (Water: n = 9; CORT: n = 9), acetyl-H3 (Water: n = 7; CORT: n = 9), phospho-ERK 1 and phospho-ERK 2 (Water: n = 8; CORT: n = 9) from the LA in rats sacrificed on the last day of CORT exposure. ( D ) Representative Western blots for each protein in (C). ( E ) Mean (±SEM) immunoreactivity for GluR1, synaptophysin, and BDNF (Water: n = 9; CORT: n = 9) from area CA3 in rats sacrificed on the last day of the wash-out period. ( F ) Mean (±SEM) immunoreactivity for GluR1 (Water: n = 9; CORT: n = 8), synaptophysin (Water: n = 9; CORT: n = 9), Arc/Arg3.1 (Water: n = 9; CORT: n = 8), Egr-1 (Water: n = 9; CORT: n = 8), BDNF (Water: n = 9; CORT: n = 9), acetyl-H3 (Water: n = 9; CORT: n = 8), phospho-ERK 1 and phospho-ERK 2 (Water: n = 9; CORT: n = 8) from the LA in rats sacrificed on the last day of the wash-out period. ( G ) Representative Western blots for each protein in (F). For each experiment, protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Water group. (*) p
    Figure Legend Snippet: Chronic exposure to CORT persistently enhances the expression of memory-related IEGs and synaptically-localized proteins in the LA. ( A ) Schematic of the behavioral protocol. Rats received either Water or CORT in their drinking water (50 μg/ml) for 2 weeks. Half the rats were sacrificed at the end of CORT exposure period. The other half was titrated off the CORT (25 μg/ml, 12.5 μg/ml) and given a 2 week period of exposure to water alone (‘wash-out’) prior to being sacrificed. ( B ) Mean (±SEM) immunoreactivity for GluR1, synaptophysin and BDNF (Water: n = 9; CORT: n = 9) from area CA3 in rats sacrificed on the last day of CORT exposure. ( C ) Mean (±SEM) immunoreactivity for GluR1 (Water: n = 9; CORT: n = 9), synaptophysin (Water: n = 9; CORT: n = 9), Arc/Arg3.1 (Water: n = 9; CORT: n = 9), Egr-1 (Water: n = 9; CORT: n = 9), BDNF (Water: n = 9; CORT: n = 9), acetyl-H3 (Water: n = 7; CORT: n = 9), phospho-ERK 1 and phospho-ERK 2 (Water: n = 8; CORT: n = 9) from the LA in rats sacrificed on the last day of CORT exposure. ( D ) Representative Western blots for each protein in (C). ( E ) Mean (±SEM) immunoreactivity for GluR1, synaptophysin, and BDNF (Water: n = 9; CORT: n = 9) from area CA3 in rats sacrificed on the last day of the wash-out period. ( F ) Mean (±SEM) immunoreactivity for GluR1 (Water: n = 9; CORT: n = 8), synaptophysin (Water: n = 9; CORT: n = 9), Arc/Arg3.1 (Water: n = 9; CORT: n = 8), Egr-1 (Water: n = 9; CORT: n = 8), BDNF (Water: n = 9; CORT: n = 9), acetyl-H3 (Water: n = 9; CORT: n = 8), phospho-ERK 1 and phospho-ERK 2 (Water: n = 9; CORT: n = 8) from the LA in rats sacrificed on the last day of the wash-out period. ( G ) Representative Western blots for each protein in (F). For each experiment, protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Water group. (*) p

    Techniques Used: Expressing, Western Blot

    12) Product Images from "Dietary diindolylmethane suppresses inflammation-driven lung squamous cell carcinoma in mice"

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

    Journal: Cancer prevention research (Philadelphia, Pa.)

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

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

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

    13) Product Images from "Downregulation of WDR20 due to loss of 14q is involved in the malignant transformation of clear cell renal cell carcinoma"

    Article Title: Downregulation of WDR20 due to loss of 14q is involved in the malignant transformation of clear cell renal cell carcinoma

    Journal: Cancer Science

    doi: 10.1111/cas.12892

    WDR 20 suppresses the phosphorylation of protein kinase B ( AKT ) and ERK in renal cell carcinoma cells. At 72 h after plating, the vector, 786O_ WDR 20 cl1, and 786O_ WDR 20 cl2 (A) or the vector, 786O_ WDR 20 cl2, and 786O_ WDR 20cl2wo (B) were subjected to Western blot analysis using antibodies against the phosphorylated form of AKT (Ser473), total AKT , the phosphorylated form of ERK (Thr202/Tyr204), total ERK , and GAPDH .
    Figure Legend Snippet: WDR 20 suppresses the phosphorylation of protein kinase B ( AKT ) and ERK in renal cell carcinoma cells. At 72 h after plating, the vector, 786O_ WDR 20 cl1, and 786O_ WDR 20 cl2 (A) or the vector, 786O_ WDR 20 cl2, and 786O_ WDR 20cl2wo (B) were subjected to Western blot analysis using antibodies against the phosphorylated form of AKT (Ser473), total AKT , the phosphorylated form of ERK (Thr202/Tyr204), total ERK , and GAPDH .

    Techniques Used: Plasmid Preparation, Western Blot

    14) Product Images from "TMEFF2 modulates the AKT and ERK signaling pathways"

    Article Title: TMEFF2 modulates the AKT and ERK signaling pathways

    Journal: International Journal of Biochemistry and Molecular Biology

    doi:

    Model of the role of TMEFF2 in Akt and ERK activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned
    Figure Legend Snippet: Model of the role of TMEFF2 in Akt and ERK activation. A: full length TMEFF2 acting as a receptor (green bars) or co-receptor (grey and green) promotes ERK phosphorylation; B: shedding of TMEFF2 leads to ectodomain accumulation (green circle) in the conditioned

    Techniques Used: Activation Assay

    The TMEFF2 ectodomain promotes AKT phosphorylation and that inversely correlates with its effect on ERK phosphorylation. RWPE-1 cells transferred to basal KSF medium for 4 hours before the medium was replaced with ectodomain containing conditioned medium
    Figure Legend Snippet: The TMEFF2 ectodomain promotes AKT phosphorylation and that inversely correlates with its effect on ERK phosphorylation. RWPE-1 cells transferred to basal KSF medium for 4 hours before the medium was replaced with ectodomain containing conditioned medium

    Techniques Used:

    Conditioned medium from HEK293T cells expressing the ectodomain construct contains secreted TMEFF2 ectodomain and inhibits ERK phosphorylation. A: Exponentially growing HEK293T cells transfected with the TMEFF2-ectodomain construct or the empty vector
    Figure Legend Snippet: Conditioned medium from HEK293T cells expressing the ectodomain construct contains secreted TMEFF2 ectodomain and inhibits ERK phosphorylation. A: Exponentially growing HEK293T cells transfected with the TMEFF2-ectodomain construct or the empty vector

    Techniques Used: Expressing, Construct, Transfection, Plasmid Preparation

    PDGF induces sustained phosphorylation of ERK in cells expressing TMEFF2. A: RWPE-1 cells were transferred to basal KSF medium for 30 minutes and then treated with various concentrations of PDGF-AA or 50 ng/ml of EGF. Whole cell lysates were then subjected
    Figure Legend Snippet: PDGF induces sustained phosphorylation of ERK in cells expressing TMEFF2. A: RWPE-1 cells were transferred to basal KSF medium for 30 minutes and then treated with various concentrations of PDGF-AA or 50 ng/ml of EGF. Whole cell lysates were then subjected

    Techniques Used: Expressing

    15) Product Images from "The BRAFV600E inhibitor, PLX4032, increases type I collagen synthesis in melanoma cells"

    Article Title: The BRAFV600E inhibitor, PLX4032, increases type I collagen synthesis in melanoma cells

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2015.05.007

    MAPK inhibition induces collagen synthesis in human melanoma cells. C4 or VMM12 cells were plated on 6-well plates at a density of 0.5 × 10 5 cells per well and treated the following day with 0.3 µl/ml DMSO, 3 µM PLX4032 (PLX) or 10 µM U0126 for 48 h. (A) RT-PCR of Col1A2 expression in mRNA from C4 cells. Fold change was calculated relative to cells treated with DMSO. Immunoblots probing for (B) pERK and total ERK (6 µg protein) and (C) Col1A2 and a non-specific band (22 µg protein) in C4 cell lysates. Pre-pro-collagen (upper band) and pro-collagen (lower band) are found between 150 kD–100 kD (after 1 min exposure), the non-specific band was found at 37 kD (after 15 min exposure). (D) RT-PCR of MMP-1 in cDNA from C4 cells. Fold change was calculated relative to cells treated with PLX. Expression of mRNA in VMM12 cells, (E) Col1A2 and (F) MMP-1, fold change was calculated as above. (G) Immunoblots of pERK and total ERK (top two panels — 6 µg protein) in VMM12 cell lysates, and (bottom panel) immunoblot of MMP-1 in VMM12 cell supernatants from the same serum-free culture (30 µl of TCA precipitate). One-way ANOVAs were performed on the relative fold changes compared to DMSO controls, data were pooled from 3 separate experiments (****P
    Figure Legend Snippet: MAPK inhibition induces collagen synthesis in human melanoma cells. C4 or VMM12 cells were plated on 6-well plates at a density of 0.5 × 10 5 cells per well and treated the following day with 0.3 µl/ml DMSO, 3 µM PLX4032 (PLX) or 10 µM U0126 for 48 h. (A) RT-PCR of Col1A2 expression in mRNA from C4 cells. Fold change was calculated relative to cells treated with DMSO. Immunoblots probing for (B) pERK and total ERK (6 µg protein) and (C) Col1A2 and a non-specific band (22 µg protein) in C4 cell lysates. Pre-pro-collagen (upper band) and pro-collagen (lower band) are found between 150 kD–100 kD (after 1 min exposure), the non-specific band was found at 37 kD (after 15 min exposure). (D) RT-PCR of MMP-1 in cDNA from C4 cells. Fold change was calculated relative to cells treated with PLX. Expression of mRNA in VMM12 cells, (E) Col1A2 and (F) MMP-1, fold change was calculated as above. (G) Immunoblots of pERK and total ERK (top two panels — 6 µg protein) in VMM12 cell lysates, and (bottom panel) immunoblot of MMP-1 in VMM12 cell supernatants from the same serum-free culture (30 µl of TCA precipitate). One-way ANOVAs were performed on the relative fold changes compared to DMSO controls, data were pooled from 3 separate experiments (****P

    Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

    Potential cross-talk between MAPK and TGFβ signaling. C4 cells in DMEM/10% FBS were treated with 3 µM PLX4032 (PLX), 2 ng/ml exogenous TGFβ, combination of PLX + TGFβ, or combination of PLX + TGFβ + 10 µMSB431542 (SB) for 0 (Ct), 15, 45, or 90 min. Cell lysates were harvested at each time point. Immunoblot analyses of (A—top two blots—6 µg protein) pERK and total ERK, (B—bottom two blots—6 µg protein) pAKT and total AKT, (C—top two blots—44 µg protein) pSmad 2/3 and total Smad 2/3, and (B—bottom blot—44 µg protein) Smad 7. (A) Samples from each blot/row were run on the same gel. (B) Samples were run on different gels (Ct, PLX, and TGFβ on one, the rest of the samples on another gel), but the gels were loaded with the same amount of protein, processed simultaneously, and treated and exposed the same way.
    Figure Legend Snippet: Potential cross-talk between MAPK and TGFβ signaling. C4 cells in DMEM/10% FBS were treated with 3 µM PLX4032 (PLX), 2 ng/ml exogenous TGFβ, combination of PLX + TGFβ, or combination of PLX + TGFβ + 10 µMSB431542 (SB) for 0 (Ct), 15, 45, or 90 min. Cell lysates were harvested at each time point. Immunoblot analyses of (A—top two blots—6 µg protein) pERK and total ERK, (B—bottom two blots—6 µg protein) pAKT and total AKT, (C—top two blots—44 µg protein) pSmad 2/3 and total Smad 2/3, and (B—bottom blot—44 µg protein) Smad 7. (A) Samples from each blot/row were run on the same gel. (B) Samples were run on different gels (Ct, PLX, and TGFβ on one, the rest of the samples on another gel), but the gels were loaded with the same amount of protein, processed simultaneously, and treated and exposed the same way.

    Techniques Used:

    16) Product Images from "PAK1 confers chemoresistance and poor outcome in non-small cell lung cancer via β-catenin-mediated stemness"

    Article Title: PAK1 confers chemoresistance and poor outcome in non-small cell lung cancer via β-catenin-mediated stemness

    Journal: Scientific Reports

    doi: 10.1038/srep34933

    MEK/ERK signaling plays a more important role thanPI3K/AKT signaling in mediating PAK1-mediated cisplatin resistance. ( a ) Six lung cancer cell types were treated with four concentrations of cisplatin and the dose response curves were used to calculate the 50% inhibition concentration (IC50) for cisplatin. The expression of PAK1 and its active forms (pS144-PAK1 and pT423-PAK1) in each lung cancer cell type were examined by western blotting. ( b ) Increasing amounts of PAK1 knockdown plasmid were transfected into high-PAK1 expressing (H441 and H23) cell lines. Alternatively, increasing amounts of expression plasmid were transfected into low PAK1 expressing (H358 and H1355) cell lines. The total amount of transfected DNA was kept constant by adding the control vector. After 48 hr, cell lysates were harvested and evaluated by Western blotting for levels of PAK1 and β-actin protein. β-actin was used as a protein loading control. NC: non-specific shRNA control. VC: Vector control. PAK1-knockdown or PAK1-overexpressing lung cancer cells were treated with four doses of cisplatin and the dose response curves were used to calculate the 50% inhibition concentration (IC50). ( c ) PAK1-overexpressing H358 and H1355 cells were treated for 5 h with inhibitors of PI3K/AKT (10 μM perifosine) and ERK (10 μM AZD6244). The inhibitors were then removed and the cells were treated with 25 μM cisplatin for an additional 48 h. Cell viability was evaluated with the MTT assay. ( d ) H23 and PAK1-overexpressing H1355 cells were transfected with β-catenin overexpression plasmid for 24 h. The cells were then treated with AZD6244 for 5 h, the inhibitor was removed, and the cells were treated with 25 μM cisplatin for an additional 48 h. Cell viability was evaluated with the MTT assay. ( e ) H23 and PAK1-overexpressing H1355 cells were treated with AZD6244 for 5 h, followed by treatment with MG132 for an additional 5 h, and then the cell lysates were evaluated for protein expression by western blotting. All experiments were performed three independent times. The mean values and the standard deviations are indicated as columns with error bars . The samples were derived from the same experiment and gels/blots were processed in parallel. Full-length blots are presented in Supplementary Figures S2 and S3 .
    Figure Legend Snippet: MEK/ERK signaling plays a more important role thanPI3K/AKT signaling in mediating PAK1-mediated cisplatin resistance. ( a ) Six lung cancer cell types were treated with four concentrations of cisplatin and the dose response curves were used to calculate the 50% inhibition concentration (IC50) for cisplatin. The expression of PAK1 and its active forms (pS144-PAK1 and pT423-PAK1) in each lung cancer cell type were examined by western blotting. ( b ) Increasing amounts of PAK1 knockdown plasmid were transfected into high-PAK1 expressing (H441 and H23) cell lines. Alternatively, increasing amounts of expression plasmid were transfected into low PAK1 expressing (H358 and H1355) cell lines. The total amount of transfected DNA was kept constant by adding the control vector. After 48 hr, cell lysates were harvested and evaluated by Western blotting for levels of PAK1 and β-actin protein. β-actin was used as a protein loading control. NC: non-specific shRNA control. VC: Vector control. PAK1-knockdown or PAK1-overexpressing lung cancer cells were treated with four doses of cisplatin and the dose response curves were used to calculate the 50% inhibition concentration (IC50). ( c ) PAK1-overexpressing H358 and H1355 cells were treated for 5 h with inhibitors of PI3K/AKT (10 μM perifosine) and ERK (10 μM AZD6244). The inhibitors were then removed and the cells were treated with 25 μM cisplatin for an additional 48 h. Cell viability was evaluated with the MTT assay. ( d ) H23 and PAK1-overexpressing H1355 cells were transfected with β-catenin overexpression plasmid for 24 h. The cells were then treated with AZD6244 for 5 h, the inhibitor was removed, and the cells were treated with 25 μM cisplatin for an additional 48 h. Cell viability was evaluated with the MTT assay. ( e ) H23 and PAK1-overexpressing H1355 cells were treated with AZD6244 for 5 h, followed by treatment with MG132 for an additional 5 h, and then the cell lysates were evaluated for protein expression by western blotting. All experiments were performed three independent times. The mean values and the standard deviations are indicated as columns with error bars . The samples were derived from the same experiment and gels/blots were processed in parallel. Full-length blots are presented in Supplementary Figures S2 and S3 .

    Techniques Used: Inhibition, Concentration Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, shRNA, MTT Assay, Over Expression, Derivative Assay

    17) Product Images from "Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway"

    Article Title: Metformin synergistically enhances antitumor activity of cisplatin in gallbladder cancer via the PI3K/AKT/ERK pathway

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0160-x

    Met potentiated the anti-tumor activity of Cis through PI3K/AKT/ERK pathway. a Expression of PI3K, p-PI3K, AKT, p-AKT, ERK and p-ERK was analyzed by Western blotting assay. Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was used as the sample loading control. For one experiment, three assays were carried out, but only one set of gels is shown. b The expression levels of PI3K, p-PI3K, AKT, p-AKT, ERK and p-ERK protein in every group were analyzed by densitometry normalized to GAPDH density c Effects of IGF-1 on Cis + Met induced apoptosis detected by AnnexinV-FITC/PI staining assay. Apoptotic cells were measured after treatment with Cis + Met in the presence or absence of IGF-1 (100 ng/mL) for 36 h. The values represent the mean ± standard deviation of three independent experiments. * P
    Figure Legend Snippet: Met potentiated the anti-tumor activity of Cis through PI3K/AKT/ERK pathway. a Expression of PI3K, p-PI3K, AKT, p-AKT, ERK and p-ERK was analyzed by Western blotting assay. Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was used as the sample loading control. For one experiment, three assays were carried out, but only one set of gels is shown. b The expression levels of PI3K, p-PI3K, AKT, p-AKT, ERK and p-ERK protein in every group were analyzed by densitometry normalized to GAPDH density c Effects of IGF-1 on Cis + Met induced apoptosis detected by AnnexinV-FITC/PI staining assay. Apoptotic cells were measured after treatment with Cis + Met in the presence or absence of IGF-1 (100 ng/mL) for 36 h. The values represent the mean ± standard deviation of three independent experiments. * P

    Techniques Used: Activity Assay, Expressing, Western Blot, Staining, Standard Deviation

    18) Product Images from "NF1-RAC1 axis regulates migration of the melanocytic lineage"

    Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2020.100858

    Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1 +/− melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1 +/− melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1 +/− melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1 -specific siRNA (si NF1 ). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1 +/− melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1 +/− melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1 -specific siRNA (si NF1 ) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. si NF1 , #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). ** P
    Figure Legend Snippet: Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1 +/− melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1 +/− melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1 +/− melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1 -specific siRNA (si NF1 ). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1 +/− melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1 +/− melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1 -specific siRNA (si NF1 ) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. si NF1 , #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). ** P

    Techniques Used: Migration, Activity Assay, Transfection, Expressing, Western Blot

    19) Product Images from "A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop"

    Article Title: A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop

    Journal: Theranostics

    doi: 10.7150/thno.22048

    The miR-630/YAP1/ERK feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of p-AKT, total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P
    Figure Legend Snippet: The miR-630/YAP1/ERK feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of p-AKT, total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Over Expression, Staining, Flow Cytometry, Cytometry, Western Blot

    MiR-630 expression levels are associated with gefitinib resistance and upregulation of miR-630 expression by gefitinib may occur by decreased SP1 binding to the miR-630 promoter due to ERK inactivation in PC9 cells, but not in PC9GR cells. (A) Six lung adenocarcinoma cells were treated with four concentrations of gefitinib. After 24 h, the dose-response curves, determined by the MTT assay, were used to calculate the IC50 values of these cells. MiR-630 expression of these cells was evaluated by real-time PCR. The expression of p-AKT, total AKT, p-ERK, total ERK and β-actin was evaluated by western blotting. (B) MiR-630 inhibitors were transfected into PC9 cells. MiR-630 mimic was transfected into low miR-630 expressing PC9GR cells. After 24 h, the cells were treated with four concentrations of gefitinib to calculate the IC50 values. NC: nonspecific shRNA control. VC: Vector control. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to NC (*P
    Figure Legend Snippet: MiR-630 expression levels are associated with gefitinib resistance and upregulation of miR-630 expression by gefitinib may occur by decreased SP1 binding to the miR-630 promoter due to ERK inactivation in PC9 cells, but not in PC9GR cells. (A) Six lung adenocarcinoma cells were treated with four concentrations of gefitinib. After 24 h, the dose-response curves, determined by the MTT assay, were used to calculate the IC50 values of these cells. MiR-630 expression of these cells was evaluated by real-time PCR. The expression of p-AKT, total AKT, p-ERK, total ERK and β-actin was evaluated by western blotting. (B) MiR-630 inhibitors were transfected into PC9 cells. MiR-630 mimic was transfected into low miR-630 expressing PC9GR cells. After 24 h, the cells were treated with four concentrations of gefitinib to calculate the IC50 values. NC: nonspecific shRNA control. VC: Vector control. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to NC (*P

    Techniques Used: Expressing, Binding Assay, MTT Assay, Real-time Polymerase Chain Reaction, Western Blot, Transfection, shRNA, Plasmid Preparation

    20) Product Images from "Granulocyte colony-stimulating factor receptor signalling via Janus kinase 2/signal transducer and activator of transcription 3 in ovarian cancer"

    Article Title: Granulocyte colony-stimulating factor receptor signalling via Janus kinase 2/signal transducer and activator of transcription 3 in ovarian cancer

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.673

    Effects of chemotherapeutic agents on downstream pathways. Cell lysates were prepared from OVCA429, HEY and TOV21G cells treated with cisplatin (Cis) or paclitaxel (Pac) as indicated, and subjected to western blot analysis with anti-phospho-STAT3, anti-STAT3 and anti-GAPDH ( A ), anti-phospho-ERK, anti-ERK, anti-phospho-AKT, anti-AKT and anti-GAPDH ( B ), or anti-MDR1, anti-GST-pi, anti-ERCC1 and anti-GAPDH ( C ).
    Figure Legend Snippet: Effects of chemotherapeutic agents on downstream pathways. Cell lysates were prepared from OVCA429, HEY and TOV21G cells treated with cisplatin (Cis) or paclitaxel (Pac) as indicated, and subjected to western blot analysis with anti-phospho-STAT3, anti-STAT3 and anti-GAPDH ( A ), anti-phospho-ERK, anti-ERK, anti-phospho-AKT, anti-AKT and anti-GAPDH ( B ), or anti-MDR1, anti-GST-pi, anti-ERCC1 and anti-GAPDH ( C ).

    Techniques Used: Western Blot

    G-CSFR signals via JAK2/STAT3 to mediate its effects on ovarian cancer cells. ( A ) Activation of STAT3 by G-CSF. Lysates from either untreated (Ctrl) cells or those treated with G-CSF or IL-6 were immunoprecipitated with anti-STAT3 and analysed by western blot with anti-phospho-STAT3 and anti-STAT3 antibodies. The input lysate was analysed using anti-GAPDH to confirm equivalent amounts of protein was examined in each case. Experiments were performed thrice and blots are representative of one experiment. ( B ) G-CSF-induced STAT3 activation is blocked by specific inhibitors of the JAK2/STAT3 pathway. Cell lysates were prepared from cells either untreated or treated with G-CSF or IL-6 with or without specific inhibitors for STAT3 (S3I) or JAK2 (J2I), as indicated, and analysed for STAT3 phosphorylation as per panel ( A ). ( C – D ) G-CSF-induced ERK activation and its inhibition by a JAK2 inhibitor. Cell lysates were prepared from untreated (Ctrl) cells or those treated with G-CSF or IL-6, and analysed by western blot with anti-phospho-ERK, anti-ERK, anti-phospho-AKT, anti-AKT and anti-GAPDH ( C ). This analysis was repeated for OVCA429 cells in the presence of JAK2 inhibitor (J2I) ( D ). ( E ) Migration is blocked by specific inhibitors of the JAK2/STAT3 pathway. Wound closure in OVCA429 cells by G-CSF and IL-6, with or without specific inhibitors for STAT3 or JAK2, as indicated (* P
    Figure Legend Snippet: G-CSFR signals via JAK2/STAT3 to mediate its effects on ovarian cancer cells. ( A ) Activation of STAT3 by G-CSF. Lysates from either untreated (Ctrl) cells or those treated with G-CSF or IL-6 were immunoprecipitated with anti-STAT3 and analysed by western blot with anti-phospho-STAT3 and anti-STAT3 antibodies. The input lysate was analysed using anti-GAPDH to confirm equivalent amounts of protein was examined in each case. Experiments were performed thrice and blots are representative of one experiment. ( B ) G-CSF-induced STAT3 activation is blocked by specific inhibitors of the JAK2/STAT3 pathway. Cell lysates were prepared from cells either untreated or treated with G-CSF or IL-6 with or without specific inhibitors for STAT3 (S3I) or JAK2 (J2I), as indicated, and analysed for STAT3 phosphorylation as per panel ( A ). ( C – D ) G-CSF-induced ERK activation and its inhibition by a JAK2 inhibitor. Cell lysates were prepared from untreated (Ctrl) cells or those treated with G-CSF or IL-6, and analysed by western blot with anti-phospho-ERK, anti-ERK, anti-phospho-AKT, anti-AKT and anti-GAPDH ( C ). This analysis was repeated for OVCA429 cells in the presence of JAK2 inhibitor (J2I) ( D ). ( E ) Migration is blocked by specific inhibitors of the JAK2/STAT3 pathway. Wound closure in OVCA429 cells by G-CSF and IL-6, with or without specific inhibitors for STAT3 or JAK2, as indicated (* P

    Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Inhibition, Migration

    21) Product Images from "Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells"

    Article Title: Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131603

    Shorter peptides containing the FGD sequence bind to OB1 B cells but fail to elicit a response. (A) Sequence of peptides nested on the FGD epitope bearing 17, 12, 8, and 4 amino acids, the mutant (mut) AGA 17-mer, and 17-mer peptides where the flanking residues were either substituted by same-polarity amino acids (17-mer subs), by hydrophobic amino acids with the exception of four serines (17-mer hydroph), or 17-mer peptides in which the core FGD was mutated to AGD or FGA. (B) OB1 B cells were stimulated with 0.5 µM of each FGD 17-mer, OVA, 12-, 8-, and 4-mer peptides for 1 and 3 min, and AGA 17-mer (mut) for 3 min, and then lysates were run in SDS-PAGE and immunoblotted with anti–phospho-ERK (top) or anti–total ERK (bottom). (C) OB1 B cells were loaded with Indo-1 AM, stimulated with 0.5 µM FGD 17-, 12-, 8-, and 4-mer and 17-mer AGA (mut), and Ca 2+ flux traces were collected. (D) ELISA plates were coated with 0.1 µM OVA, and then 50 ng/ml of whole OB1 antibody (left) or 500 ng/ml OB1 antibody Fab (right) plus increasing concentrations of FGD 17-, 12-, 8-, and 4-mer, AGA 17-mer mutant peptide, or OVA were added. Plates were developed with anti-IgG1 HRP (left) or anti-Fab HRP (right). (E) Competitive ELISA was performed as in D but adding increasing concentrations of 17-mer subs, hydroph, AGD, or FGA, according to the sequences in A. Results are representative of three (B and C) independent experiments. In D and E, mean + SEM of two (whole antibody) and three (Fab) independent experiments is shown.
    Figure Legend Snippet: Shorter peptides containing the FGD sequence bind to OB1 B cells but fail to elicit a response. (A) Sequence of peptides nested on the FGD epitope bearing 17, 12, 8, and 4 amino acids, the mutant (mut) AGA 17-mer, and 17-mer peptides where the flanking residues were either substituted by same-polarity amino acids (17-mer subs), by hydrophobic amino acids with the exception of four serines (17-mer hydroph), or 17-mer peptides in which the core FGD was mutated to AGD or FGA. (B) OB1 B cells were stimulated with 0.5 µM of each FGD 17-mer, OVA, 12-, 8-, and 4-mer peptides for 1 and 3 min, and AGA 17-mer (mut) for 3 min, and then lysates were run in SDS-PAGE and immunoblotted with anti–phospho-ERK (top) or anti–total ERK (bottom). (C) OB1 B cells were loaded with Indo-1 AM, stimulated with 0.5 µM FGD 17-, 12-, 8-, and 4-mer and 17-mer AGA (mut), and Ca 2+ flux traces were collected. (D) ELISA plates were coated with 0.1 µM OVA, and then 50 ng/ml of whole OB1 antibody (left) or 500 ng/ml OB1 antibody Fab (right) plus increasing concentrations of FGD 17-, 12-, 8-, and 4-mer, AGA 17-mer mutant peptide, or OVA were added. Plates were developed with anti-IgG1 HRP (left) or anti-Fab HRP (right). (E) Competitive ELISA was performed as in D but adding increasing concentrations of 17-mer subs, hydroph, AGD, or FGA, according to the sequences in A. Results are representative of three (B and C) independent experiments. In D and E, mean + SEM of two (whole antibody) and three (Fab) independent experiments is shown.

    Techniques Used: Sequencing, Mutagenesis, SDS Page, Enzyme-linked Immunosorbent Assay, Competitive ELISA

    OVA-specific OB1 B cells are activated by peptide containing the epitope FGD. (A) OB1 B cells were incubated on ice in the absence (control) or presence of biotinylated versions of 0.5 µM FGD 17-mer and AGA 17-mer peptides. Binding was detected by flow cytometry using SA-APC. (B) OB1 B cells were stimulated with equimolar (0.5 µM) FGD 17-mer, AGA 17-mer, or OVA or 5 µg/ml anti-IgG1 or anti-IgM for 3 min, and then lysates were run in SDS-PAGE and immunoblotted with anti–phospho-ERK (top) or anti–total ERK (bottom). (C) OB1 B cells were loaded with Ca 2+ -sensitive Indo-1 AM dye and collected for 1 min (unstimulated baseline). After that, 5 µM FGD 17-mer, AGA 17-mer, or OVA or 5 µg/ml anti-IgG1 was added and collected for five additional minutes. (D) OB1 B cells were resuspended in serum-free or serum-complete RPMI, and Ca 2+ trace was collected as in C upon incubation with 0.5 µM FGD 17-mer. (E) OB1 B cells loaded with Indo-1 AM dye were preincubated with 50 nM Btk inhibitor for 30 min at 37°C, and Ca 2+ trace was collected upon stimulation with 0.5 µM FGD 17-mer. (F) Render of z-stacks of confocal microscopy of OB1 MHC II GFP B cells that were incubated for 10 min with Alexa Fluor 647–labeled FGD or AGA 17-mer or FluB1 MHC II B cells stimulated with Alexa Fluor 647–labeled FGD 17-mer. (G) OB1 B cells were either left unstimulated or stimulated with 17-mer or anti-IgG for 15 min in the presence of 100 µM dynasore at 37°C and stained with anti–mouse IgG antibody conjugated with Alexa Fluor 405 and Alexa Fluor 647 for STORM imaging. Representative fields (left) and single-cell images from selected box regions (right) are shown. Bars: (F) 2 µm; (G, left) 5 µm; (G, right) 1 µm. (H) The percentage of clusters that have more than three BCRs (top), the cluster size (middle), and the number of localizations within clusters (bottom) between unstimulated, 17-mer, and anti-IgG conditions were quantified, and means and SDs are plotted. Unpaired Student’s t test was performed comparing the total number of clusters contained in unstimulated versus the 17-mer or the anti-IgG condition. The number of cells analyzed was 67 (unstimulated), 102 (17-mer), and 118 (anti-IgG). P values of resulting analysis are shown: *, P = 0.024; ***, P = 0.0004; ****, P
    Figure Legend Snippet: OVA-specific OB1 B cells are activated by peptide containing the epitope FGD. (A) OB1 B cells were incubated on ice in the absence (control) or presence of biotinylated versions of 0.5 µM FGD 17-mer and AGA 17-mer peptides. Binding was detected by flow cytometry using SA-APC. (B) OB1 B cells were stimulated with equimolar (0.5 µM) FGD 17-mer, AGA 17-mer, or OVA or 5 µg/ml anti-IgG1 or anti-IgM for 3 min, and then lysates were run in SDS-PAGE and immunoblotted with anti–phospho-ERK (top) or anti–total ERK (bottom). (C) OB1 B cells were loaded with Ca 2+ -sensitive Indo-1 AM dye and collected for 1 min (unstimulated baseline). After that, 5 µM FGD 17-mer, AGA 17-mer, or OVA or 5 µg/ml anti-IgG1 was added and collected for five additional minutes. (D) OB1 B cells were resuspended in serum-free or serum-complete RPMI, and Ca 2+ trace was collected as in C upon incubation with 0.5 µM FGD 17-mer. (E) OB1 B cells loaded with Indo-1 AM dye were preincubated with 50 nM Btk inhibitor for 30 min at 37°C, and Ca 2+ trace was collected upon stimulation with 0.5 µM FGD 17-mer. (F) Render of z-stacks of confocal microscopy of OB1 MHC II GFP B cells that were incubated for 10 min with Alexa Fluor 647–labeled FGD or AGA 17-mer or FluB1 MHC II B cells stimulated with Alexa Fluor 647–labeled FGD 17-mer. (G) OB1 B cells were either left unstimulated or stimulated with 17-mer or anti-IgG for 15 min in the presence of 100 µM dynasore at 37°C and stained with anti–mouse IgG antibody conjugated with Alexa Fluor 405 and Alexa Fluor 647 for STORM imaging. Representative fields (left) and single-cell images from selected box regions (right) are shown. Bars: (F) 2 µm; (G, left) 5 µm; (G, right) 1 µm. (H) The percentage of clusters that have more than three BCRs (top), the cluster size (middle), and the number of localizations within clusters (bottom) between unstimulated, 17-mer, and anti-IgG conditions were quantified, and means and SDs are plotted. Unpaired Student’s t test was performed comparing the total number of clusters contained in unstimulated versus the 17-mer or the anti-IgG condition. The number of cells analyzed was 67 (unstimulated), 102 (17-mer), and 118 (anti-IgG). P values of resulting analysis are shown: *, P = 0.024; ***, P = 0.0004; ****, P

    Techniques Used: Incubation, Binding Assay, Flow Cytometry, Cytometry, SDS Page, Confocal Microscopy, Labeling, Staining, Imaging

    22) Product Images from "Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori"

    Article Title: Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00043

    Time- (A) and dose- (B) dependent effects of JNK phosphorylation by the PTTH, tissue specificity of PTTH-stimulated JNK phosphorylation (C) , and the effect of treatment with extracellular fluid from cells only or cells infected with the wild-type (WT) AcMNPV on protein phosphorylation (D) . (A,B) Time- and dose-dependent effects. PGs were either treated with PTTH for the indicated time points (A) , treated with the indicated concentrations of PTTH, or incubated with control medium (0) for 60 min (B) . (C) Tissue specificity. PGs, subesophageal ganglia (SoG), wing disks (WD), salivary glands (SG), and fat body (FB) from day 6-last instar larvae were incubated with control medium (CN) or medium containing the PTTH (P) for 60 min. (D) Effect of treatment with either control medium (CN), the PTTH (P), extracellular fluid from cells only (EF), or cells infected with WT AcMNPV (WT) on the phosphorylation of JNK, ERK, and 4E-BP. Each lysate was prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-phospho-ERK (P-ERK), anti-phospho-4E-BP (P-4E-BP), anti-JNK (JNK), and anti-α-tubulin (α-tubulin) antibodies. Results shown in the left panels are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A–C ) or α-tubulin (for D ). Asterisks indicate a significant difference compared to the respective control (by Student's t -test, ** p
    Figure Legend Snippet: Time- (A) and dose- (B) dependent effects of JNK phosphorylation by the PTTH, tissue specificity of PTTH-stimulated JNK phosphorylation (C) , and the effect of treatment with extracellular fluid from cells only or cells infected with the wild-type (WT) AcMNPV on protein phosphorylation (D) . (A,B) Time- and dose-dependent effects. PGs were either treated with PTTH for the indicated time points (A) , treated with the indicated concentrations of PTTH, or incubated with control medium (0) for 60 min (B) . (C) Tissue specificity. PGs, subesophageal ganglia (SoG), wing disks (WD), salivary glands (SG), and fat body (FB) from day 6-last instar larvae were incubated with control medium (CN) or medium containing the PTTH (P) for 60 min. (D) Effect of treatment with either control medium (CN), the PTTH (P), extracellular fluid from cells only (EF), or cells infected with WT AcMNPV (WT) on the phosphorylation of JNK, ERK, and 4E-BP. Each lysate was prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-phospho-ERK (P-ERK), anti-phospho-4E-BP (P-4E-BP), anti-JNK (JNK), and anti-α-tubulin (α-tubulin) antibodies. Results shown in the left panels are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A–C ) or α-tubulin (for D ). Asterisks indicate a significant difference compared to the respective control (by Student's t -test, ** p

    Techniques Used: Infection, Incubation

    Effects of SP600125 on PTTH-stimulated phosphorylation of JNK (A) and ERK (B) and ecdysteroid secretion (C) . PGs were pretreated with 20 μM SP600125 or vehicle alone for 30 min, and then transferred to medium containing the same dose of SP600125, with or without the PTTH. Incubation was maintained for 60 min. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-JNK (JNK), anti-phospho-ERK (P-ERK), and anti-ERK (ERK) antibodies. CN, glands incubated in control medium; S, glands incubated in medium containing 20 μM SP600125 only; P, glands incubated in medium containing the PTTH only; P+S, glands incubated in medium containing both the PTTH and 20 μM SP600125. Results shown in the left panels (for A,B ) are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A ) or ERK (for B ). Ecdysteroid released into the medium was determined by an RIA. Different letters above the bars indicate a significant difference (ANOVA followed by Tukey's multiple-comparisons test, p
    Figure Legend Snippet: Effects of SP600125 on PTTH-stimulated phosphorylation of JNK (A) and ERK (B) and ecdysteroid secretion (C) . PGs were pretreated with 20 μM SP600125 or vehicle alone for 30 min, and then transferred to medium containing the same dose of SP600125, with or without the PTTH. Incubation was maintained for 60 min. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-JNK (JNK), anti-phospho-ERK (P-ERK), and anti-ERK (ERK) antibodies. CN, glands incubated in control medium; S, glands incubated in medium containing 20 μM SP600125 only; P, glands incubated in medium containing the PTTH only; P+S, glands incubated in medium containing both the PTTH and 20 μM SP600125. Results shown in the left panels (for A,B ) are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A ) or ERK (for B ). Ecdysteroid released into the medium was determined by an RIA. Different letters above the bars indicate a significant difference (ANOVA followed by Tukey's multiple-comparisons test, p

    Techniques Used: Incubation

    23) Product Images from "Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori"

    Article Title: Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00043

    Time- (A) and dose- (B) dependent effects of JNK phosphorylation by the PTTH, tissue specificity of PTTH-stimulated JNK phosphorylation (C) , and the effect of treatment with extracellular fluid from cells only or cells infected with the wild-type (WT) AcMNPV on protein phosphorylation (D) . (A,B) Time- and dose-dependent effects. PGs were either treated with PTTH for the indicated time points (A) , treated with the indicated concentrations of PTTH, or incubated with control medium (0) for 60 min (B) . (C) Tissue specificity. PGs, subesophageal ganglia (SoG), wing disks (WD), salivary glands (SG), and fat body (FB) from day 6-last instar larvae were incubated with control medium (CN) or medium containing the PTTH (P) for 60 min. (D) Effect of treatment with either control medium (CN), the PTTH (P), extracellular fluid from cells only (EF), or cells infected with WT AcMNPV (WT) on the phosphorylation of JNK, ERK, and 4E-BP. Each lysate was prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-phospho-ERK (P-ERK), anti-phospho-4E-BP (P-4E-BP), anti-JNK (JNK), and anti-α-tubulin (α-tubulin) antibodies. Results shown in the left panels are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A–C ) or α-tubulin (for D ). Asterisks indicate a significant difference compared to the respective control (by Student's t -test, ** p
    Figure Legend Snippet: Time- (A) and dose- (B) dependent effects of JNK phosphorylation by the PTTH, tissue specificity of PTTH-stimulated JNK phosphorylation (C) , and the effect of treatment with extracellular fluid from cells only or cells infected with the wild-type (WT) AcMNPV on protein phosphorylation (D) . (A,B) Time- and dose-dependent effects. PGs were either treated with PTTH for the indicated time points (A) , treated with the indicated concentrations of PTTH, or incubated with control medium (0) for 60 min (B) . (C) Tissue specificity. PGs, subesophageal ganglia (SoG), wing disks (WD), salivary glands (SG), and fat body (FB) from day 6-last instar larvae were incubated with control medium (CN) or medium containing the PTTH (P) for 60 min. (D) Effect of treatment with either control medium (CN), the PTTH (P), extracellular fluid from cells only (EF), or cells infected with WT AcMNPV (WT) on the phosphorylation of JNK, ERK, and 4E-BP. Each lysate was prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-phospho-ERK (P-ERK), anti-phospho-4E-BP (P-4E-BP), anti-JNK (JNK), and anti-α-tubulin (α-tubulin) antibodies. Results shown in the left panels are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A–C ) or α-tubulin (for D ). Asterisks indicate a significant difference compared to the respective control (by Student's t -test, ** p

    Techniques Used: Infection, Incubation

    Effects of SP600125 on PTTH-stimulated phosphorylation of JNK (A) and ERK (B) and ecdysteroid secretion (C) . PGs were pretreated with 20 μM SP600125 or vehicle alone for 30 min, and then transferred to medium containing the same dose of SP600125, with or without the PTTH. Incubation was maintained for 60 min. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-JNK (JNK), anti-phospho-ERK (P-ERK), and anti-ERK (ERK) antibodies. CN, glands incubated in control medium; S, glands incubated in medium containing 20 μM SP600125 only; P, glands incubated in medium containing the PTTH only; P+S, glands incubated in medium containing both the PTTH and 20 μM SP600125. Results shown in the left panels (for A,B ) are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A ) or ERK (for B ). Ecdysteroid released into the medium was determined by an RIA. Different letters above the bars indicate a significant difference (ANOVA followed by Tukey's multiple-comparisons test, p
    Figure Legend Snippet: Effects of SP600125 on PTTH-stimulated phosphorylation of JNK (A) and ERK (B) and ecdysteroid secretion (C) . PGs were pretreated with 20 μM SP600125 or vehicle alone for 30 min, and then transferred to medium containing the same dose of SP600125, with or without the PTTH. Incubation was maintained for 60 min. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-JNK (P-JNK), anti-JNK (JNK), anti-phospho-ERK (P-ERK), and anti-ERK (ERK) antibodies. CN, glands incubated in control medium; S, glands incubated in medium containing 20 μM SP600125 only; P, glands incubated in medium containing the PTTH only; P+S, glands incubated in medium containing both the PTTH and 20 μM SP600125. Results shown in the left panels (for A,B ) are representative of three independent experiments. Data are expressed as multiples of change over the respective control after being normalized to the level of JNK (for A ) or ERK (for B ). Ecdysteroid released into the medium was determined by an RIA. Different letters above the bars indicate a significant difference (ANOVA followed by Tukey's multiple-comparisons test, p

    Techniques Used: Incubation

    24) Product Images from "Effects of Chronic Sleep Deprivation on the Extracellular Signal-Regulated Kinase Pathway in the Temporomandibular Joint of Rats"

    Article Title: Effects of Chronic Sleep Deprivation on the Extracellular Signal-Regulated Kinase Pathway in the Temporomandibular Joint of Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107544

    Expression levels of p-ERK, ERK, MMP-1, MMP-3, and MMP-13 in the condyles. Western blot technique was used to examine the possible mechanism by which pathological alterations occur in the TMJ. (A) Comparison of the p-ERK, ERK, MMP-1, MMP-3, and MMP-13 protein levels in the different groups as determined by Western blot (WB). (B) Mean relative protein levels of p-ERK, ERK, MMP-1, MMP-3, and MMP-13 in different groups (n = 10 per group). Bars represent the mean and SD of each group. CON, control; CSD, chronic sleep deprivation; d, day. ** P
    Figure Legend Snippet: Expression levels of p-ERK, ERK, MMP-1, MMP-3, and MMP-13 in the condyles. Western blot technique was used to examine the possible mechanism by which pathological alterations occur in the TMJ. (A) Comparison of the p-ERK, ERK, MMP-1, MMP-3, and MMP-13 protein levels in the different groups as determined by Western blot (WB). (B) Mean relative protein levels of p-ERK, ERK, MMP-1, MMP-3, and MMP-13 in different groups (n = 10 per group). Bars represent the mean and SD of each group. CON, control; CSD, chronic sleep deprivation; d, day. ** P

    Techniques Used: Expressing, Western Blot

    25) Product Images from "Increasing Dietary Selenium Elevates Reducing Capacity and ERK Activation Associated with Accelerated Progression of Select Mesothelioma Tumors"

    Article Title: Increasing Dietary Selenium Elevates Reducing Capacity and ERK Activation Associated with Accelerated Progression of Select Mesothelioma Tumors

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.12.008

    The effects of selenium on the proliferation and mobility of MM cell lines are mediated by ERK activation. A: Western blot analysis on cell lysates from 1-week-old cells show an increase in pERK levels with increasing selenium [low (30 nmol/L), medium
    Figure Legend Snippet: The effects of selenium on the proliferation and mobility of MM cell lines are mediated by ERK activation. A: Western blot analysis on cell lysates from 1-week-old cells show an increase in pERK levels with increasing selenium [low (30 nmol/L), medium

    Techniques Used: Activation Assay, Western Blot

    26) Product Images from "Hepatic Growth Hormone Resistance After Acute Injury"

    Article Title: Hepatic Growth Hormone Resistance After Acute Injury

    Journal: Endocrinology

    doi: 10.1210/en.2012-2134

    Effect of trauma alone and combined trauma and hemorrhage for 90 minutes on hepatic GH-induced JAK/STAT signaling. Mice were subjected to trauma alone and injected with GH or saline immediately (T0′), 90 minutes later (T90′), or after 90 minutes hemorrhage (TH90′), and livers were harvested 10 minutes later. A, Western analysis of liver lysates was performed, and blots were probed with specific anti-PY694/699-STAT5, anti-STAT5, and anti-ERK antibodies. Representative lanes from the scanned image of a single film were chosen; bands of interest were cropped and rearranged to enhance clarity. ERK was used to demonstrate even loading. B, Bands of interest from Western blot autoradiographs were quantified by scanning densitometry, and differences in STAT5 phosphorylation between GH+ groups were determined. C, Individual P-STAT5 bands were quantified by scanning densitometry, and the ratios of the upper vs lower bands were calculated. D, The same liver lysates as in A were immunoprecipitated with anti-JAK2 antibody. JAK2 immunoprecipitates were subjected to Western analysis and probed with specific anti-PY1007/1008-JAK2 and anti-JAK2 antibodies. E, Bands of interest were quantified by scanning densitometry, and differences in JAK2 phosphorylation between GH+ groups were determined. Except for C, data are presented as mean ± SEM percent change in phosphorylation, normalized to total protein. P-STAT5 after T0′ was arbitrarily set to 100%. * P
    Figure Legend Snippet: Effect of trauma alone and combined trauma and hemorrhage for 90 minutes on hepatic GH-induced JAK/STAT signaling. Mice were subjected to trauma alone and injected with GH or saline immediately (T0′), 90 minutes later (T90′), or after 90 minutes hemorrhage (TH90′), and livers were harvested 10 minutes later. A, Western analysis of liver lysates was performed, and blots were probed with specific anti-PY694/699-STAT5, anti-STAT5, and anti-ERK antibodies. Representative lanes from the scanned image of a single film were chosen; bands of interest were cropped and rearranged to enhance clarity. ERK was used to demonstrate even loading. B, Bands of interest from Western blot autoradiographs were quantified by scanning densitometry, and differences in STAT5 phosphorylation between GH+ groups were determined. C, Individual P-STAT5 bands were quantified by scanning densitometry, and the ratios of the upper vs lower bands were calculated. D, The same liver lysates as in A were immunoprecipitated with anti-JAK2 antibody. JAK2 immunoprecipitates were subjected to Western analysis and probed with specific anti-PY1007/1008-JAK2 and anti-JAK2 antibodies. E, Bands of interest were quantified by scanning densitometry, and differences in JAK2 phosphorylation between GH+ groups were determined. Except for C, data are presented as mean ± SEM percent change in phosphorylation, normalized to total protein. P-STAT5 after T0′ was arbitrarily set to 100%. * P

    Techniques Used: Mouse Assay, Injection, Western Blot, Immunoprecipitation

    Effects of soft-tissue trauma on GH-induced STAT5 tyrosine phosphorylation. Mice were subjected to either laparotomy alone or surgical trauma (laparotomy, wound closure, and catheterization), and GH or saline was immediately injected into the inferior vena cava. Livers were removed 10 minutes later. A, Western analysis of liver lysates was performed, and blots were probed with specific anti-PY694/699-STAT5, anti-STAT5, and anti-ERK antibodies. Representative lanes from the scanned image of a single film were chosen; bands of interest were cropped and rearranged to enhance clarity. ERK was used to control for loading on separate P-STAT5 and T-STAT5 gels. B, Bands of interest were quantified by scanning densitometry, and differences in STAT5 phosphorylation between groups were determined. Data are presented as mean ± SEM percent change in phosphorylation, normalized to total STAT5. T0′ GH+ was arbitrarily set to 100%. * P
    Figure Legend Snippet: Effects of soft-tissue trauma on GH-induced STAT5 tyrosine phosphorylation. Mice were subjected to either laparotomy alone or surgical trauma (laparotomy, wound closure, and catheterization), and GH or saline was immediately injected into the inferior vena cava. Livers were removed 10 minutes later. A, Western analysis of liver lysates was performed, and blots were probed with specific anti-PY694/699-STAT5, anti-STAT5, and anti-ERK antibodies. Representative lanes from the scanned image of a single film were chosen; bands of interest were cropped and rearranged to enhance clarity. ERK was used to control for loading on separate P-STAT5 and T-STAT5 gels. B, Bands of interest were quantified by scanning densitometry, and differences in STAT5 phosphorylation between groups were determined. Data are presented as mean ± SEM percent change in phosphorylation, normalized to total STAT5. T0′ GH+ was arbitrarily set to 100%. * P

    Techniques Used: Mouse Assay, Injection, Western Blot

    27) Product Images from "Overexpression of cGMP-dependent protein kinase I (PKG-I) attenuates ischemia-reperfusion-induced kidney injury"

    Article Title: Overexpression of cGMP-dependent protein kinase I (PKG-I) attenuates ischemia-reperfusion-induced kidney injury

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00355.2011

    Expression of Bcl-2, Bag-1, and p-ERK in the kidneys from sham or IR groups of mice. Expression of antiapoptotic genes (Bcl-2 and Bag-1) in kidney from 4 groups of mice was analyzed by real-time PCR ( A ) and immunoblotting ( B ). Values are means ±
    Figure Legend Snippet: Expression of Bcl-2, Bag-1, and p-ERK in the kidneys from sham or IR groups of mice. Expression of antiapoptotic genes (Bcl-2 and Bag-1) in kidney from 4 groups of mice was analyzed by real-time PCR ( A ) and immunoblotting ( B ). Values are means ±

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    28) Product Images from "Age and Tissue Specific Differences in the Development of Acute Insulin Resistance Following Injury"

    Article Title: Age and Tissue Specific Differences in the Development of Acute Insulin Resistance Following Injury

    Journal: The Journal of endocrinology

    doi: 10.1677/JOE-09-0269

    Decreasing corticosterone levels did not affect trauma and hemorrhage-induced insulin resistance in liver Metyrapone (Met) was administered as described in Materials and Methods to 6- or 10-week old rats which were then subjected to trauma, or trauma and hemorrhage. Liver tissue lysates were subjected to Western blotting with antibodies specific for P-Akt (S473), P-IR (Y972)], P-IRS1 (Y612), T-Akt, and T-ERK. A, a representative Western blot. B, C, D, autoradiographs were quantified by scanning densitometry and presented as the mean ± SEM of three rats in each group and presented as the percentage change by insulin with the 10-week old rats arbitrarily set to 100%.
    Figure Legend Snippet: Decreasing corticosterone levels did not affect trauma and hemorrhage-induced insulin resistance in liver Metyrapone (Met) was administered as described in Materials and Methods to 6- or 10-week old rats which were then subjected to trauma, or trauma and hemorrhage. Liver tissue lysates were subjected to Western blotting with antibodies specific for P-Akt (S473), P-IR (Y972)], P-IRS1 (Y612), T-Akt, and T-ERK. A, a representative Western blot. B, C, D, autoradiographs were quantified by scanning densitometry and presented as the mean ± SEM of three rats in each group and presented as the percentage change by insulin with the 10-week old rats arbitrarily set to 100%.

    Techniques Used: Western Blot

    Hepatic and skeletal muscle insulin signaling via serine phosphorylation of Akt at different ages following trauma and hemorrhage Rats were subjected to trauma alone (T90’) or trauma and hemorrhage (TH90’). At 90’ (with or without hemorrhage) either saline (−) or 5U insulin (Ins; +) was injected via the portal vein. The liver and triceps were removed after 1 min and frozen in liquid nitrogen. A and C, tissue lysates from liver and triceps were subjected to Western blotting with antibodies specific for phospho-Akt [P-Akt (S473)], total Akt (T-Akt), and total ERK (T-ERK). B and D, autoradiographs were quantified by scanning densitometry. Data are presented as the mean ± SEM of the percentage change of P-Akt (S473) by insulin of three rats in each group. The phosphorylated protein levels in T90’ (3 weeks) with insulin treatment were arbitrarily set to 100 percent. In this and all the following figures, *, p
    Figure Legend Snippet: Hepatic and skeletal muscle insulin signaling via serine phosphorylation of Akt at different ages following trauma and hemorrhage Rats were subjected to trauma alone (T90’) or trauma and hemorrhage (TH90’). At 90’ (with or without hemorrhage) either saline (−) or 5U insulin (Ins; +) was injected via the portal vein. The liver and triceps were removed after 1 min and frozen in liquid nitrogen. A and C, tissue lysates from liver and triceps were subjected to Western blotting with antibodies specific for phospho-Akt [P-Akt (S473)], total Akt (T-Akt), and total ERK (T-ERK). B and D, autoradiographs were quantified by scanning densitometry. Data are presented as the mean ± SEM of the percentage change of P-Akt (S473) by insulin of three rats in each group. The phosphorylated protein levels in T90’ (3 weeks) with insulin treatment were arbitrarily set to 100 percent. In this and all the following figures, *, p

    Techniques Used: Injection, Western Blot

    29) Product Images from "Role of Tissue Macrophages in the Development of Critical Illness Diabetes"

    Article Title: Role of Tissue Macrophages in the Development of Critical Illness Diabetes

    Journal: Shock (Augusta, Ga.)

    doi: 10.1097/SHK.0b013e31823180a4

    Effect of macrophage depletion by GdCl 3 on the activation of JNK and IKK kinases in liver following trauma and hemorrhage Rats were injected with GdCl 3 (10 mg/kg) via the tail vein, at 48 hours post-injection, surgical trauma and hemorrhage for 90 min was performed and liver tissues were harvested. (A). Liver protein extracts were subjected to Western Blot analysis with anti-PT181/Y183-JNK1/2, PS181-IKKβ/PS180-IKKα, and total-ERK antibodies. (B, C, D). The levels of P-JNK1 (B), P-IKKα (C) and P-IKKβ (D) in liver was presented as the mean +/− SEM in the graph (n=4–9 for each group; TH90’: trauma and hemorrhage for 90 min; T90’: trauma alone for 90 min).
    Figure Legend Snippet: Effect of macrophage depletion by GdCl 3 on the activation of JNK and IKK kinases in liver following trauma and hemorrhage Rats were injected with GdCl 3 (10 mg/kg) via the tail vein, at 48 hours post-injection, surgical trauma and hemorrhage for 90 min was performed and liver tissues were harvested. (A). Liver protein extracts were subjected to Western Blot analysis with anti-PT181/Y183-JNK1/2, PS181-IKKβ/PS180-IKKα, and total-ERK antibodies. (B, C, D). The levels of P-JNK1 (B), P-IKKα (C) and P-IKKβ (D) in liver was presented as the mean +/− SEM in the graph (n=4–9 for each group; TH90’: trauma and hemorrhage for 90 min; T90’: trauma alone for 90 min).

    Techniques Used: Activation Assay, Injection, Western Blot

    30) Product Images from "Inactivation of DAP12 in PMN Inhibits TREM1-Mediated Activation in Rheumatoid Arthritis"

    Article Title: Inactivation of DAP12 in PMN Inhibits TREM1-Mediated Activation in Rheumatoid Arthritis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115116

    Increased activation of PMN cells from RA patients. (A) Primary RA PMN were incubated with pre-fixed (1% paraformaldehyde) HTB-93 cells and collected at 0, 5, 15, 30 minutes and the lysate was run on a western blot and probed for the activity of ERK and AKT or total ERK/AKT as a loading control. (B) PMN cells isolated from the synovial fluid of RA patients and healthy blood donors were used in a cytotoxic assay with 51 Cr-labeled HTB-93 cells. Percentage specific lysis of HTB-93 cells was compared using at effector to target (E:T) ratios of 50:1, 25:1,12.5:1, and 6.25:1. The mean and SD of triplicate determinations are shown.
    Figure Legend Snippet: Increased activation of PMN cells from RA patients. (A) Primary RA PMN were incubated with pre-fixed (1% paraformaldehyde) HTB-93 cells and collected at 0, 5, 15, 30 minutes and the lysate was run on a western blot and probed for the activity of ERK and AKT or total ERK/AKT as a loading control. (B) PMN cells isolated from the synovial fluid of RA patients and healthy blood donors were used in a cytotoxic assay with 51 Cr-labeled HTB-93 cells. Percentage specific lysis of HTB-93 cells was compared using at effector to target (E:T) ratios of 50:1, 25:1,12.5:1, and 6.25:1. The mean and SD of triplicate determinations are shown.

    Techniques Used: Activation Assay, Incubation, Western Blot, Activity Assay, Isolation, Labeling, Lysis

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    Western Blot:

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    Article Snippet: .. The following antibodies were used in Western blot analyses and immunocytochemistry: rabbit anti‐TNF‐α (ab6671), goat anti‐rabbit FITC‐conjugated IgG H & L (ab6717), and rabbit anti‐CCR7 (ab103404) from Abcam (Cambridge, MA, USA); rabbit anti‐p38 (8690), rabbit anti‐phospho‐p38 (Thr180/Tyr182; 4511), rabbit anti‐ERK (9102), rabbit anti‐phospho‐ERK (Thr202/Tyr204; 9101), and HRP‐conjugated anti‐rabbit IgG (7074) antibodies from Cell Signaling Technology (Danvers, MA, USA); and mouse anti‐GAPDH (NB300‐221) antibody from Novus Biologicals (Littleton, CO, USA). .. The ERK inhibitor U0126 (9903) and the p38 inhibitor SB203580 (5633) were purchased from Cell Signaling Technology.

    Article Title: High-Intensity Interval Training Improves Markers of Oxidative Metabolism in Skeletal Muscle of Individuals With Obesity and Insulin Resistance
    Article Snippet: .. For Western blot analyses, muscle lysate (approximately 50 mg cellular protein) was separated by SDS-PAGE, electro-transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States), and probed overnight with phospho-SAPK/JNK (Thr183/Tyr185, Cell Signaling, #9251), phospho-p38 (Thr180/Tyr182, Cell Signaling, #9211), phospho-ERK1/2 (Cell Signaling, #9102), phospho-Akt (Ser473, Cell Signaling, #9271), phospho-IRS-1 (Ser612-C15H5, Life Sciences, #44816), phospho-AS160 (Thr642, Cell Signaling, #8881), GLUT4 (1F8, Cell Signaling, #2213), βHAD (Proteintech, #19828-1-AP), COX IV (3E1, Cell Signaling, #4850), TFAM (Cell Signaling, #7495), PGC1α (Calbiochem, # ST1202), and GAPDH (14C10, Cell Signaling, #2118) antibodies. ..

    Immunocytochemistry:

    Article Title: Tumor necrosis factor‐α induces prostate cancer cell migration in lymphatic metastasis through CCR7 upregulation, et al. Tumor necrosis factor‐α induces prostate cancer cell migration in lymphatic metastasis through CCR7 upregulation
    Article Snippet: .. The following antibodies were used in Western blot analyses and immunocytochemistry: rabbit anti‐TNF‐α (ab6671), goat anti‐rabbit FITC‐conjugated IgG H & L (ab6717), and rabbit anti‐CCR7 (ab103404) from Abcam (Cambridge, MA, USA); rabbit anti‐p38 (8690), rabbit anti‐phospho‐p38 (Thr180/Tyr182; 4511), rabbit anti‐ERK (9102), rabbit anti‐phospho‐ERK (Thr202/Tyr204; 9101), and HRP‐conjugated anti‐rabbit IgG (7074) antibodies from Cell Signaling Technology (Danvers, MA, USA); and mouse anti‐GAPDH (NB300‐221) antibody from Novus Biologicals (Littleton, CO, USA). .. The ERK inhibitor U0126 (9903) and the p38 inhibitor SB203580 (5633) were purchased from Cell Signaling Technology.

    SDS Page:

    Article Title: High-Intensity Interval Training Improves Markers of Oxidative Metabolism in Skeletal Muscle of Individuals With Obesity and Insulin Resistance
    Article Snippet: .. For Western blot analyses, muscle lysate (approximately 50 mg cellular protein) was separated by SDS-PAGE, electro-transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States), and probed overnight with phospho-SAPK/JNK (Thr183/Tyr185, Cell Signaling, #9251), phospho-p38 (Thr180/Tyr182, Cell Signaling, #9211), phospho-ERK1/2 (Cell Signaling, #9102), phospho-Akt (Ser473, Cell Signaling, #9271), phospho-IRS-1 (Ser612-C15H5, Life Sciences, #44816), phospho-AS160 (Thr642, Cell Signaling, #8881), GLUT4 (1F8, Cell Signaling, #2213), βHAD (Proteintech, #19828-1-AP), COX IV (3E1, Cell Signaling, #4850), TFAM (Cell Signaling, #7495), PGC1α (Calbiochem, # ST1202), and GAPDH (14C10, Cell Signaling, #2118) antibodies. ..

    Activation Assay:

    Article Title: Ginsenoside F1 Promotes Cytotoxic Activity of NK Cells via Insulin-Like Growth Factor-1-Dependent Mechanism
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    Cell Signaling Technology Inc anti total extracellular signal related kinase erk
    Arachidonic acid (AA) increases the expression of growth and survival factor genes in human dermal papillary cells (hDPCs). (A) The mRNA for fibroblast growth factors (FGF-7 and -10) and hepatocyte growth factor (HGF) is elevated in hDPCs that were treated with AA (1, 2 µM, or 5 µM) for 24 hours. (B) Treatment with AA increases the expression of FGF-7 and FGF-10 in the cytoplasm of the dermal papilla (DP) and inner root sheath (IRS) on day 3 of its application, compared to the vehicle control (×100). (C and D) Human DPCs were lysed to analyze the total protein content of the lysates via Western blotting by using primary antibodies for total-p42/44 <t>ERK,</t> phospho-p42/44 ERK, <t>total-AKT,</t> phospho-AKT, total-CREB, phospho-CREB, Bcl-2, and β-actin. ERK: extracellular signal-regulated kinases, AKT: protein kinase B, CREB: cAMP response element-binding protein, CTL, control. Data are expressed as mean±standard error. * p ≤0.05, ** p ≤0.01, *** p ≤0.01, versus the control group.
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    Involvement of <t>Erk-1/2</t> and JNK in LPA induced RON expression in human bladder cancer T24 cells. ( A ) T24 cells were incubated with 5 μM LPA for various periods, and the levels of phosphorylated Erk-1/2, P38 and JNK MAPK in the cell lysates were determined by Western blot analysis. ( B ) T24 cells, after being pretreated with PD98059 (PD, 50 μM), SB203580 (SB, 10 μM), and SP600125 (SP, 10 μM) for 1 h, were incubated with 5 μM LPA for 8 h. After incubation, the RON mRNA in the cell lysates was determined by RT-PCR analysis. ( C ) T24 cells transfected with Erk-1/2 (K97M) and JNK (TAM67) dominant negative mutants for 48 h were then co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA PMA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p
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    Increased HIF-1α expression in activated B cells. a Quantitative RT-PCR analyses of Hif1a and Hif2a in wild-type (WT) splenic B cells stimulated with lipopolysaccharide (LPS) or anti-IgM at indicated time points ( n = 4 at each time point). Values at 0 h were set as 1. b Expression of HIF-1α and HIF-2α in WT splenic B cells stimulated with LPS or anti-IgM at indicated time points ( n = 3 experiments in duplicate). c Western blot and densitometry analysis of phospho-STAT3 <t>Ser727</t> (pSTAT3 727 ), phospho-STAT3 Tyr705 (pSTAT3 705 ), total STAT3, <t>phospho-ERK</t> (pERK), and total ERK in WT splenic B cells after stimulation with anti-IgM at indicated time points ( n = 3 experiments in duplicate). d Western blot and densitometry analysis of HIF-1α, pSTAT3 727 , and pERK in anti-IgM-stimulated B cells with or without STAT3, ERK, or AKT inhibitors treatments for 4 h ( n = 3 experiments in duplicate). e Scheme of Hif1a promoter indicating the potential STAT3 binding site position and enrichment of pSTAT3 727 on Hif1a promoter in splenic B cells 4 h after stimulation with anti-IgM or medium (Med) ( n = 4 for all groups). Data are shown as mean ± s.e.m. Pictures are representative of three ( a – d ) or four ( e ) independent experiments. * P
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    Arachidonic acid (AA) increases the expression of growth and survival factor genes in human dermal papillary cells (hDPCs). (A) The mRNA for fibroblast growth factors (FGF-7 and -10) and hepatocyte growth factor (HGF) is elevated in hDPCs that were treated with AA (1, 2 µM, or 5 µM) for 24 hours. (B) Treatment with AA increases the expression of FGF-7 and FGF-10 in the cytoplasm of the dermal papilla (DP) and inner root sheath (IRS) on day 3 of its application, compared to the vehicle control (×100). (C and D) Human DPCs were lysed to analyze the total protein content of the lysates via Western blotting by using primary antibodies for total-p42/44 ERK, phospho-p42/44 ERK, total-AKT, phospho-AKT, total-CREB, phospho-CREB, Bcl-2, and β-actin. ERK: extracellular signal-regulated kinases, AKT: protein kinase B, CREB: cAMP response element-binding protein, CTL, control. Data are expressed as mean±standard error. * p ≤0.05, ** p ≤0.01, *** p ≤0.01, versus the control group.

    Journal: Annals of Dermatology

    Article Title: Role of Arachidonic Acid in Promoting Hair Growth

    doi: 10.5021/ad.2016.28.1.55

    Figure Lengend Snippet: Arachidonic acid (AA) increases the expression of growth and survival factor genes in human dermal papillary cells (hDPCs). (A) The mRNA for fibroblast growth factors (FGF-7 and -10) and hepatocyte growth factor (HGF) is elevated in hDPCs that were treated with AA (1, 2 µM, or 5 µM) for 24 hours. (B) Treatment with AA increases the expression of FGF-7 and FGF-10 in the cytoplasm of the dermal papilla (DP) and inner root sheath (IRS) on day 3 of its application, compared to the vehicle control (×100). (C and D) Human DPCs were lysed to analyze the total protein content of the lysates via Western blotting by using primary antibodies for total-p42/44 ERK, phospho-p42/44 ERK, total-AKT, phospho-AKT, total-CREB, phospho-CREB, Bcl-2, and β-actin. ERK: extracellular signal-regulated kinases, AKT: protein kinase B, CREB: cAMP response element-binding protein, CTL, control. Data are expressed as mean±standard error. * p ≤0.05, ** p ≤0.01, *** p ≤0.01, versus the control group.

    Article Snippet: The blotted membranes were incubated at 4℃ with the appropriate antibodies: anti-total extracellular signal-related kinase (ERK), anti-phosphorylated ERK, anti-total protein kinase B (Akt), anti-phosphorylated AKT, anti-B-cell lymphoma 2 (Bcl-2), anti-total cyclic AMP response element-binding protein (CREB), anti-phosphorylated CREB (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin (Santa Cruz Biotechnology).

    Techniques: Expressing, Western Blot, Binding Assay, CTL Assay

    Involvement of Erk-1/2 and JNK in LPA induced RON expression in human bladder cancer T24 cells. ( A ) T24 cells were incubated with 5 μM LPA for various periods, and the levels of phosphorylated Erk-1/2, P38 and JNK MAPK in the cell lysates were determined by Western blot analysis. ( B ) T24 cells, after being pretreated with PD98059 (PD, 50 μM), SB203580 (SB, 10 μM), and SP600125 (SP, 10 μM) for 1 h, were incubated with 5 μM LPA for 8 h. After incubation, the RON mRNA in the cell lysates was determined by RT-PCR analysis. ( C ) T24 cells transfected with Erk-1/2 (K97M) and JNK (TAM67) dominant negative mutants for 48 h were then co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA PMA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Lysophosphatidic Acid Upregulates Recepteur D’origine Nantais Expression and Cell Invasion via Egr-1, AP-1, and NF-κB Signaling in Bladder Carcinoma Cells

    doi: 10.3390/ijms21010304

    Figure Lengend Snippet: Involvement of Erk-1/2 and JNK in LPA induced RON expression in human bladder cancer T24 cells. ( A ) T24 cells were incubated with 5 μM LPA for various periods, and the levels of phosphorylated Erk-1/2, P38 and JNK MAPK in the cell lysates were determined by Western blot analysis. ( B ) T24 cells, after being pretreated with PD98059 (PD, 50 μM), SB203580 (SB, 10 μM), and SP600125 (SP, 10 μM) for 1 h, were incubated with 5 μM LPA for 8 h. After incubation, the RON mRNA in the cell lysates was determined by RT-PCR analysis. ( C ) T24 cells transfected with Erk-1/2 (K97M) and JNK (TAM67) dominant negative mutants for 48 h were then co-transfected with a RON promoter reporter overnight. After incubation with 5 μM LPA PMA for 8 h, luciferase activity was measured with a luminometer. Bars show the mean standard deviation from three measurements. * p

    Article Snippet: To measure the loading quantity of samples, the blotted membrane was stripped with 62.5 mM Tris−HCl (pH 7.4) containing 2% SDS and 100 mM 2-mercaptoethanol, followed by hybridization with β-actin antibody (Santa Cruz, Dallas, Texas, USA) or antibodies against total Erk-1/2, JNK and p38MAPK (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Dominant Negative Mutation, Luciferase, Activity Assay, Standard Deviation

    The miR-630/YAP1/ERK feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of p-AKT, total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P

    Journal: Theranostics

    Article Title: A low microRNA-630 expression confers resistance to tyrosine kinase inhibitors in EGFR-mutated lung adenocarcinomas via miR-630/YAP1/ERK feedback loop

    doi: 10.7150/thno.22048

    Figure Lengend Snippet: The miR-630/YAP1/ERK feedback loop may be responsible for gefitinib resistance in EGFR-mutated lung adenocarcinoma cells. (A) The cell lysates of PC9 and PC9GR were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (B) MiR-630 inhibitor were transfected into PC9 cells. MiR-630 mimic was transfected into low PC9GR cells. After 48 h, the cells lysates were evaluated for the expression of YAP1, Bcl-2, Slug, and IGF1R by real-time PCR. (C) PC9 cells were transfected with the indicated combination of miR-630 inhibitor, shYAP1, shBcl-2, and shSlug for 24 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic, YAP1, Bcl-2, and Slug overexpression plasmids for 24 h. These cells were treated with 0.1% DMSO or 10 μM of gefitinib for 24 h and were subjected to annexin-V and PI staining, followed by a flow cytometry analysis. The percentages of apoptotic cells in the annexin V+/PI- population plus annexin-V+/PI+ are summarized. (D) PC9 cells were transfected with the indicated combination of miR-630 inhibitor and shYAP1 for 48 h. PC9GR cells were transfected with the indicated combination of miR-630 mimic and YAP1 overexpression plasmids for 48 h. The cells lysates were evaluated for expression of p-AKT, total AKT, p-ERK, total ERK and β-actin by western blotting. (E) YAP1-overexpressing PC9 and PC9GR cells were treated with 10 μM AZD6244 for 5 h. The cell lysates were evaluated for the expression of YAP1, p-ERK, total ERK and β-actin by western blotting. MiR-630 expression of these cells were evaluated by real-time PCR. P value was calculated by the Student's t -test. The significant differences in experimental groups were compared to vehicle or indicated treatment (*P

    Article Snippet: Anti-phospho-AKT (p-AKT), Anti-AKT, anti-total ERK, and anti-phospho-ERK (p-ERK) antibodies were obtained from Cell Signaling (Danvers, MA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Over Expression, Staining, Flow Cytometry, Cytometry, Western Blot

    Increased HIF-1α expression in activated B cells. a Quantitative RT-PCR analyses of Hif1a and Hif2a in wild-type (WT) splenic B cells stimulated with lipopolysaccharide (LPS) or anti-IgM at indicated time points ( n = 4 at each time point). Values at 0 h were set as 1. b Expression of HIF-1α and HIF-2α in WT splenic B cells stimulated with LPS or anti-IgM at indicated time points ( n = 3 experiments in duplicate). c Western blot and densitometry analysis of phospho-STAT3 Ser727 (pSTAT3 727 ), phospho-STAT3 Tyr705 (pSTAT3 705 ), total STAT3, phospho-ERK (pERK), and total ERK in WT splenic B cells after stimulation with anti-IgM at indicated time points ( n = 3 experiments in duplicate). d Western blot and densitometry analysis of HIF-1α, pSTAT3 727 , and pERK in anti-IgM-stimulated B cells with or without STAT3, ERK, or AKT inhibitors treatments for 4 h ( n = 3 experiments in duplicate). e Scheme of Hif1a promoter indicating the potential STAT3 binding site position and enrichment of pSTAT3 727 on Hif1a promoter in splenic B cells 4 h after stimulation with anti-IgM or medium (Med) ( n = 4 for all groups). Data are shown as mean ± s.e.m. Pictures are representative of three ( a – d ) or four ( e ) independent experiments. * P

    Journal: Nature Communications

    Article Title: Hypoxia-inducible factor-1α is a critical transcription factor for IL-10-producing B cells in autoimmune disease

    doi: 10.1038/s41467-017-02683-x

    Figure Lengend Snippet: Increased HIF-1α expression in activated B cells. a Quantitative RT-PCR analyses of Hif1a and Hif2a in wild-type (WT) splenic B cells stimulated with lipopolysaccharide (LPS) or anti-IgM at indicated time points ( n = 4 at each time point). Values at 0 h were set as 1. b Expression of HIF-1α and HIF-2α in WT splenic B cells stimulated with LPS or anti-IgM at indicated time points ( n = 3 experiments in duplicate). c Western blot and densitometry analysis of phospho-STAT3 Ser727 (pSTAT3 727 ), phospho-STAT3 Tyr705 (pSTAT3 705 ), total STAT3, phospho-ERK (pERK), and total ERK in WT splenic B cells after stimulation with anti-IgM at indicated time points ( n = 3 experiments in duplicate). d Western blot and densitometry analysis of HIF-1α, pSTAT3 727 , and pERK in anti-IgM-stimulated B cells with or without STAT3, ERK, or AKT inhibitors treatments for 4 h ( n = 3 experiments in duplicate). e Scheme of Hif1a promoter indicating the potential STAT3 binding site position and enrichment of pSTAT3 727 on Hif1a promoter in splenic B cells 4 h after stimulation with anti-IgM or medium (Med) ( n = 4 for all groups). Data are shown as mean ± s.e.m. Pictures are representative of three ( a – d ) or four ( e ) independent experiments. * P

    Article Snippet: The following primary antibodies were used (Supplementary Table ): HIF-1α antibody, HIF-2α antibody (Novus), STAT3 phosphorylated at Ser727 and Thr705 antibodies, total STAT3 antibody, phosphorylated ERK antibody, total ERK antibody (Cell Signaling), and β-actin antibody (Sigma).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay