erk inhibitor u0126  (Millipore)


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    Structured Review

    Millipore erk inhibitor u0126
    CCR7 regulates <t>ERK</t> expression in different cells. (A) Western blot analysis of pERK in OE and NC cells pretreated with or without CCL21 and <t>U0126.</t> GAPDH was used as a loading control. (B) Transcription levels of ERK1/2 in OE and NC cells with or without pretreatment with CCL21 and U0126. **P
    Erk Inhibitor U0126, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    erk inhibitor u0126 - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway"

    Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8962

    CCR7 regulates ERK expression in different cells. (A) Western blot analysis of pERK in OE and NC cells pretreated with or without CCL21 and U0126. GAPDH was used as a loading control. (B) Transcription levels of ERK1/2 in OE and NC cells with or without pretreatment with CCL21 and U0126. **P
    Figure Legend Snippet: CCR7 regulates ERK expression in different cells. (A) Western blot analysis of pERK in OE and NC cells pretreated with or without CCL21 and U0126. GAPDH was used as a loading control. (B) Transcription levels of ERK1/2 in OE and NC cells with or without pretreatment with CCL21 and U0126. **P

    Techniques Used: Expressing, Western Blot

    Effects of CCL21/CCR7 axis and the extracellular signal-regulated kinase inhibitor, U0126, on proliferation and migration. OE cells were pretreated with or without U0126 for 2 h, followed by stimulation with CCL21 for 16 h. (A) Proliferative ability of OE cells and NC cells pretreated with or without CCL21 and U0126. (B) Migratory ability of OE and NC cells pretreated with or without CCL21 and U0126. (C) Transwell migration assay was used to evaluate the migration capacity of different cells (magnification, ×100). **P
    Figure Legend Snippet: Effects of CCL21/CCR7 axis and the extracellular signal-regulated kinase inhibitor, U0126, on proliferation and migration. OE cells were pretreated with or without U0126 for 2 h, followed by stimulation with CCL21 for 16 h. (A) Proliferative ability of OE cells and NC cells pretreated with or without CCL21 and U0126. (B) Migratory ability of OE and NC cells pretreated with or without CCL21 and U0126. (C) Transwell migration assay was used to evaluate the migration capacity of different cells (magnification, ×100). **P

    Techniques Used: Migration, Transwell Migration Assay

    2) Product Images from "Paricalcitol Inhibits Aldosterone-Induced Proinflammatory Factors by Modulating Epidermal Growth Factor Receptor Pathway in Cultured Tubular Epithelial Cells"

    Article Title: Paricalcitol Inhibits Aldosterone-Induced Proinflammatory Factors by Modulating Epidermal Growth Factor Receptor Pathway in Cultured Tubular Epithelial Cells

    Journal: BioMed Research International

    doi: 10.1155/2015/783538

    ADAM17/TGF- α /EGFR signaling blockade inhibits aldosterone-mediated proinflammatory factors upregulation. Cells were pretreated with the specific ADAM17 inhibitor TAPI-2 (50 μ mol/L; 1 hour), a neutralizing antibody against TGF α (2.5 μ g/mL, 24 hour), the EGFR kinase inhibitor AG 1478 (100 nmol/L, 1 hour), or the ERK inhibitor U0126 (10 μ mol/L; 1 hour) before stimulation with 1 μ mol/L Aldo for 6 hours. CCL-2 and CCL-5 gene expression levels were determined by real-time PCR. Data are expressed as mean ± SEM of 2-3 experiments. * P
    Figure Legend Snippet: ADAM17/TGF- α /EGFR signaling blockade inhibits aldosterone-mediated proinflammatory factors upregulation. Cells were pretreated with the specific ADAM17 inhibitor TAPI-2 (50 μ mol/L; 1 hour), a neutralizing antibody against TGF α (2.5 μ g/mL, 24 hour), the EGFR kinase inhibitor AG 1478 (100 nmol/L, 1 hour), or the ERK inhibitor U0126 (10 μ mol/L; 1 hour) before stimulation with 1 μ mol/L Aldo for 6 hours. CCL-2 and CCL-5 gene expression levels were determined by real-time PCR. Data are expressed as mean ± SEM of 2-3 experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    3) Product Images from "Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung"

    Article Title: Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011322

    Expression of tight junction proteins are altered following 1 hour incubation with the ERK inhibitor U0126. Significant decreases in claudin 4, 18, and occludin are observed in DMSO control treated wells (*, p
    Figure Legend Snippet: Expression of tight junction proteins are altered following 1 hour incubation with the ERK inhibitor U0126. Significant decreases in claudin 4, 18, and occludin are observed in DMSO control treated wells (*, p

    Techniques Used: Expressing, Incubation

    Transepithelial resistance of sham and 2CLP monolayers following treatment with either U0126 or SP600125 to inhibit ERK or JNK activation respectively. Monolayers were serum deprived for 2 hours prior to the application of the inhibitor (time = 0). Measurements were taken at 1, 1.5, and 2 hours following treatment. 2CLP monolayers have significantly lower TER values than sham, and sham monolayer TER was unaffected by any of the treatments. Only U0126 significantly improved TER in 2CLP monolayers above initial values by 1.5 hours, and this persisted to 2 hours (*, p
    Figure Legend Snippet: Transepithelial resistance of sham and 2CLP monolayers following treatment with either U0126 or SP600125 to inhibit ERK or JNK activation respectively. Monolayers were serum deprived for 2 hours prior to the application of the inhibitor (time = 0). Measurements were taken at 1, 1.5, and 2 hours following treatment. 2CLP monolayers have significantly lower TER values than sham, and sham monolayer TER was unaffected by any of the treatments. Only U0126 significantly improved TER in 2CLP monolayers above initial values by 1.5 hours, and this persisted to 2 hours (*, p

    Techniques Used: Activation Assay

    Immunofluorescent staining of the tight junction proteins occluding, ZO-1, claudin 4, and claudin 18 in 2CLP and sham monolayers treated for 1 hour with the ERK inhibitor U0126 or DMSO control. Scale bar 50 µm.
    Figure Legend Snippet: Immunofluorescent staining of the tight junction proteins occluding, ZO-1, claudin 4, and claudin 18 in 2CLP and sham monolayers treated for 1 hour with the ERK inhibitor U0126 or DMSO control. Scale bar 50 µm.

    Techniques Used: Staining

    4) Product Images from "Intracellular Ca2+ homeostasis and JAK1/STAT3 pathway are involved in the protective effect of propofol on BV2 microglia against hypoxia-induced inflammation and apoptosis"

    Article Title: Intracellular Ca2+ homeostasis and JAK1/STAT3 pathway are involved in the protective effect of propofol on BV2 microglia against hypoxia-induced inflammation and apoptosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0178098

    Effects of propofol, BAPTA-AM, KN93, U0126 on CoCl 2 induced the phosphorylation of CAMKIIα, ERK, NF-κB. CoCl 2 -induced the phosphorylation of CAMKIIα, ERK and NF-κB were attenuated by propofol, BAPTA-AM, KN93 and U0126. (* p
    Figure Legend Snippet: Effects of propofol, BAPTA-AM, KN93, U0126 on CoCl 2 induced the phosphorylation of CAMKIIα, ERK, NF-κB. CoCl 2 -induced the phosphorylation of CAMKIIα, ERK and NF-κB were attenuated by propofol, BAPTA-AM, KN93 and U0126. (* p

    Techniques Used:

    5) Product Images from "4-1BB Controls Regulatory Activity in Dendritic Cells through Promoting Optimal Expression of Retinal Dehydrogenase"

    Article Title: 4-1BB Controls Regulatory Activity in Dendritic Cells through Promoting Optimal Expression of Retinal Dehydrogenase

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

    doi: 10.4049/jimmunol.1201248

    4-1BB is a signaling receptor for RALDH induction in DC A, MLN DC, isolated by MACS, were cultured for 1 or 2 days in the absence of stimulation with control IgG, or stimulated with an agonist antibody to 4-1BB (25 μg/ml). Activity of RALDH was assayed by ALDEFLUOR staining. Percentages of ALDEFLUOR+ cells are indicated. B–F , MACS- isolated SDC were stimulated with GM-CSF and Zymosan for 48 h. Where indicated, agonist anti-4-1BB (25 μg/ml) or rat IgG was added at the start of culture. B , 4-1BB expression in SDC stimulated with GM-CSF (100 pg/ml) and Zymosan (25 μg/ml). C , Activity of RALDH in SDC was determined by ALDEFLUOR staining when treated with GM-CSF (10 pg/ml), Zymosan (2.5 μg/ml), and Pam-3-cys (1 μg/ml) for 48h. D – E , SDC, pre-treated with Zymosan in the presence of IgG or agonist anti-4-1BB for 48h, were cultured with naïve OT-II CD4 T cells and OVA peptide (0.01 μM) for 4 days. Where indicated, TGF-β (5 ng/ml) and LE540, the pan antagonist of RA receptor, (2 μM) were added. Treg/Th1 development was determined by intracellular staining after restimulation of cells with PMA and ionomycin. Representative flow plot ( D ) and % Foxp3+ T cells in individual cultures ( E ) are shown. F , Inhibitors for signaling pathways were added at the start of culture when SDC were stimulated with Zymosan (25 μg/ml). ERK, U0126 (5 μM); PI3K, Ly (5 μM); NFκB (1 μM); Wnt/β-Catenin (5 μM). Results are representative of three experiments.
    Figure Legend Snippet: 4-1BB is a signaling receptor for RALDH induction in DC A, MLN DC, isolated by MACS, were cultured for 1 or 2 days in the absence of stimulation with control IgG, or stimulated with an agonist antibody to 4-1BB (25 μg/ml). Activity of RALDH was assayed by ALDEFLUOR staining. Percentages of ALDEFLUOR+ cells are indicated. B–F , MACS- isolated SDC were stimulated with GM-CSF and Zymosan for 48 h. Where indicated, agonist anti-4-1BB (25 μg/ml) or rat IgG was added at the start of culture. B , 4-1BB expression in SDC stimulated with GM-CSF (100 pg/ml) and Zymosan (25 μg/ml). C , Activity of RALDH in SDC was determined by ALDEFLUOR staining when treated with GM-CSF (10 pg/ml), Zymosan (2.5 μg/ml), and Pam-3-cys (1 μg/ml) for 48h. D – E , SDC, pre-treated with Zymosan in the presence of IgG or agonist anti-4-1BB for 48h, were cultured with naïve OT-II CD4 T cells and OVA peptide (0.01 μM) for 4 days. Where indicated, TGF-β (5 ng/ml) and LE540, the pan antagonist of RA receptor, (2 μM) were added. Treg/Th1 development was determined by intracellular staining after restimulation of cells with PMA and ionomycin. Representative flow plot ( D ) and % Foxp3+ T cells in individual cultures ( E ) are shown. F , Inhibitors for signaling pathways were added at the start of culture when SDC were stimulated with Zymosan (25 μg/ml). ERK, U0126 (5 μM); PI3K, Ly (5 μM); NFκB (1 μM); Wnt/β-Catenin (5 μM). Results are representative of three experiments.

    Techniques Used: Isolation, Magnetic Cell Separation, Cell Culture, Activity Assay, Staining, Expressing, Flow Cytometry

    6) Product Images from "[6]-Gingerol Prevents Disassembly of Cell Junctions and Activities of MMPs in Invasive Human Pancreas Cancer Cells through ERK/NF-κB/Snail Signal Transduction Pathway"

    Article Title: [6]-Gingerol Prevents Disassembly of Cell Junctions and Activities of MMPs in Invasive Human Pancreas Cancer Cells through ERK/NF-κB/Snail Signal Transduction Pathway

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/761852

    Effect of [6]-gingerol on the DNA-binding activity of NF- κ B in PANC-1 cells. (a) Nuclear and cytosolic extracts were prepared using the NE-PER nuclear and cytoplasmic extraction kit from PANC-1 cells treated with [6]-gingerol for 24 h. Western blotting was then performed on each fraction. Actin was used as an internal control for, the cytosolic fraction, and lamin was applied as an internal control for the nuclear fraction. (b) Cells cultured under the same conditions were pretreated for 30 min with U0126, an ERK inhibitor. Cells were then tested for the DNA-binding activity of NF- κ B by EMSA. Band intensities in the immunoblots were quantified by densitometry using L Process and MultiGauge software. Band intensities were normalized relative to the internal control and background. Data represent the mean ± SD from three independent experiments. Statistical analyses (Student's t -test and one-way ANOVA) were performed using GraphPad Prism 5. * P
    Figure Legend Snippet: Effect of [6]-gingerol on the DNA-binding activity of NF- κ B in PANC-1 cells. (a) Nuclear and cytosolic extracts were prepared using the NE-PER nuclear and cytoplasmic extraction kit from PANC-1 cells treated with [6]-gingerol for 24 h. Western blotting was then performed on each fraction. Actin was used as an internal control for, the cytosolic fraction, and lamin was applied as an internal control for the nuclear fraction. (b) Cells cultured under the same conditions were pretreated for 30 min with U0126, an ERK inhibitor. Cells were then tested for the DNA-binding activity of NF- κ B by EMSA. Band intensities in the immunoblots were quantified by densitometry using L Process and MultiGauge software. Band intensities were normalized relative to the internal control and background. Data represent the mean ± SD from three independent experiments. Statistical analyses (Student's t -test and one-way ANOVA) were performed using GraphPad Prism 5. * P

    Techniques Used: Binding Assay, Activity Assay, Western Blot, Cell Culture, Software

    7) Product Images from "TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts"

    Article Title: TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202323

    Inhibition of ERK pathway modulates UVA-induced MMP-1 and IL-8 production in UVA-irradiated senescent HDFs. (A) Senescent HDFs were treated overnight with different concentrations of U0126 (ERK inhibitor) and irradiated with UVA (1 J/cm 2 ). Cells were cultured for 4 h and protein levels of phosphorylated/total ERK were analyzed by western blotting. Anti-β-tubulin monoclonal antibody was used as a loading control. The images shown are representative of three independent experiments. (B) Total cellular ROS levels were determined by measuring DCF-DA levels using a spectrofluorometer. % ROS production in response to UVA is shown in the graph. Data are presented as percentage compared to the control (DMSO-treated cells). (C) MMP-1 and (D) IL-8 secretion levels in the culture supernatant were measured by ELISA. The values are mean ± SEM of three different experiments. *p
    Figure Legend Snippet: Inhibition of ERK pathway modulates UVA-induced MMP-1 and IL-8 production in UVA-irradiated senescent HDFs. (A) Senescent HDFs were treated overnight with different concentrations of U0126 (ERK inhibitor) and irradiated with UVA (1 J/cm 2 ). Cells were cultured for 4 h and protein levels of phosphorylated/total ERK were analyzed by western blotting. Anti-β-tubulin monoclonal antibody was used as a loading control. The images shown are representative of three independent experiments. (B) Total cellular ROS levels were determined by measuring DCF-DA levels using a spectrofluorometer. % ROS production in response to UVA is shown in the graph. Data are presented as percentage compared to the control (DMSO-treated cells). (C) MMP-1 and (D) IL-8 secretion levels in the culture supernatant were measured by ELISA. The values are mean ± SEM of three different experiments. *p

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

    8) Product Images from "Reactive Oxygen Species Hydrogen Peroxide Mediates Kaposi's Sarcoma-Associated Herpesvirus Reactivation from Latency"

    Article Title: Reactive Oxygen Species Hydrogen Peroxide Mediates Kaposi's Sarcoma-Associated Herpesvirus Reactivation from Latency

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002054

    H 2 O 2 induction of KSHV reactivation is mediated by ERK1/2, JNK, and p38 MAPK pathways. (A) Treatment with H 2 O 2 , ATZ or TPA activated ERK1/2, JNK and p38 pathways and their downstream transcriptional factor c-Jun, and induced the expression of RTA protein in BCBL1 cells harboring BAC36. Cells were treated with different concentrations of H 2 O 2 and ATZ, or TPA at 20 ng/ml with DMSO control or inhibitors of MAPK pathways including 10 µM U0126 (ERK inhibitor), 50 µM JNK inhibitor II, and 50 µM SB203580 (p38 inhibitor) for 12 h. Total ERK1/2, JNK, p38 andc-Jun, and their phosphorylated forms, RTA protein, and β-tubulin were detected by Western-blotting. (B–C) Inhibitors of MAPK pathways inhibited the induction of RTA and production of infectious virions by H 2 O 2 , ATZ and TPA in BCBL1 cells harboring BAC36. H 2 O 2 , ATZ and TPA were used at concentrations of 300 µM, 5 mM and 20 ng/ml, respectively. Inhibitors of MAPK pathways were used at concentrations described in (A). Relative RTA transcript level at 24 h of treatment was detected by RT-qPCR with untreated cells set as “1” (B). Relative virus titers were determined by using supernatants collected at 5 days of treatment to infect endothelial cells and calculating the numbers of GFP-positive cells at 48 hpi (C). Virus titers from untreated cells were set as “1”. (D) H 2 O 2 and ATZ, and TPA at concentrations of 300 µM, 5 mM and 20 ng/ml, respectively, induced the expression of RTA transcript in 293T cells harboring BAC36. RTA transcript was detected by RT-qPCR following 24 h of treatment. (E) Dominant negative (DN) constructs of MAPK pathways inhibited the induction of RTA transcript by H 2 O 2 and TPA in 293T cells. 293T cells harboring BAC36 were transiently transfected with the control plasmid and DN constructs of ERK, JNK, p38, and c-Jun for 24 h, and treated with H 2 O 2 at 300 µM and TPA at 20 ng/ml for an additional 12 h.
    Figure Legend Snippet: H 2 O 2 induction of KSHV reactivation is mediated by ERK1/2, JNK, and p38 MAPK pathways. (A) Treatment with H 2 O 2 , ATZ or TPA activated ERK1/2, JNK and p38 pathways and their downstream transcriptional factor c-Jun, and induced the expression of RTA protein in BCBL1 cells harboring BAC36. Cells were treated with different concentrations of H 2 O 2 and ATZ, or TPA at 20 ng/ml with DMSO control or inhibitors of MAPK pathways including 10 µM U0126 (ERK inhibitor), 50 µM JNK inhibitor II, and 50 µM SB203580 (p38 inhibitor) for 12 h. Total ERK1/2, JNK, p38 andc-Jun, and their phosphorylated forms, RTA protein, and β-tubulin were detected by Western-blotting. (B–C) Inhibitors of MAPK pathways inhibited the induction of RTA and production of infectious virions by H 2 O 2 , ATZ and TPA in BCBL1 cells harboring BAC36. H 2 O 2 , ATZ and TPA were used at concentrations of 300 µM, 5 mM and 20 ng/ml, respectively. Inhibitors of MAPK pathways were used at concentrations described in (A). Relative RTA transcript level at 24 h of treatment was detected by RT-qPCR with untreated cells set as “1” (B). Relative virus titers were determined by using supernatants collected at 5 days of treatment to infect endothelial cells and calculating the numbers of GFP-positive cells at 48 hpi (C). Virus titers from untreated cells were set as “1”. (D) H 2 O 2 and ATZ, and TPA at concentrations of 300 µM, 5 mM and 20 ng/ml, respectively, induced the expression of RTA transcript in 293T cells harboring BAC36. RTA transcript was detected by RT-qPCR following 24 h of treatment. (E) Dominant negative (DN) constructs of MAPK pathways inhibited the induction of RTA transcript by H 2 O 2 and TPA in 293T cells. 293T cells harboring BAC36 were transiently transfected with the control plasmid and DN constructs of ERK, JNK, p38, and c-Jun for 24 h, and treated with H 2 O 2 at 300 µM and TPA at 20 ng/ml for an additional 12 h.

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Dominant Negative Mutation, Construct, Transfection, Plasmid Preparation

    9) Product Images from "Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor"

    Article Title: Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2017.1312045

    Upregulation of IL-21R on the NK cell surface is facilitated via the MAP kinase signaling pathway. (A) NK cells were stimulated with PMA or left resting, with the addition or absence of the ERK inhibitor U0126 and subjected to immunoblot analysis for phospho-ERK expression. (B) NK cells were stimulated for 8 hr with immobilized-IgG or left unstimulated in the presence of the ERK inhibitor U0126 (reconstituted in DMSO) and subjected to immunoblot analysis for IL-21R expression. The membranes were re-probed for β-actin to confirm equal loading. NK cells were isolated from two healthy donors.
    Figure Legend Snippet: Upregulation of IL-21R on the NK cell surface is facilitated via the MAP kinase signaling pathway. (A) NK cells were stimulated with PMA or left resting, with the addition or absence of the ERK inhibitor U0126 and subjected to immunoblot analysis for phospho-ERK expression. (B) NK cells were stimulated for 8 hr with immobilized-IgG or left unstimulated in the presence of the ERK inhibitor U0126 (reconstituted in DMSO) and subjected to immunoblot analysis for IL-21R expression. The membranes were re-probed for β-actin to confirm equal loading. NK cells were isolated from two healthy donors.

    Techniques Used: Expressing, Isolation

    10) Product Images from "Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain"

    Article Title: Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39472-z

    U0126 reversed acid-induced mechanical allodynia in the sham but not OVX rats. ( A ) Intrathecal (i.t.) injection of the ERK inhibitor U0126 (10 μg in 20 μL 5% DMSO) in the ovary-intact (Sham) rats on day 5 after the 2nd AI (PD5) attenuated mechanical allodynia at 3 h and the next day (PD6). This effect was not detected in vehicle-treated rats (Sham-DMSO). ( B ) No difference was observed in OVX rats with i.t. U0126 or vehicle injection. BL, before 1st injection; PD1, day 1 post 2nd AI; PD5-3 h, 3 h post U0126/or vehicle injection; PD6, day 6 post 2nd AI. ## p
    Figure Legend Snippet: U0126 reversed acid-induced mechanical allodynia in the sham but not OVX rats. ( A ) Intrathecal (i.t.) injection of the ERK inhibitor U0126 (10 μg in 20 μL 5% DMSO) in the ovary-intact (Sham) rats on day 5 after the 2nd AI (PD5) attenuated mechanical allodynia at 3 h and the next day (PD6). This effect was not detected in vehicle-treated rats (Sham-DMSO). ( B ) No difference was observed in OVX rats with i.t. U0126 or vehicle injection. BL, before 1st injection; PD1, day 1 post 2nd AI; PD5-3 h, 3 h post U0126/or vehicle injection; PD6, day 6 post 2nd AI. ## p

    Techniques Used: Injection

    Immunofluorescence analysis indicates the effect of ERK inhibitor on acid injection-induced ERK activation in the spinal dorsal horn. U0126 or vehicle (doses as Fig. 7 ) was injected 5 days after the 2nd AI. ( A ) Immunofluorescence reveals the p-ERK distribution in the L4/5 ipsilateral spinal cords before the 1st AI (Pre) and 3 h post U0126 or DMSO injection. Upper panels: Sham; lower panels: OVX. Scale bar: 100 μm. ( B ) Comparisons of p-ERK-ir cell numbers among the sham and OVX groups with DMSO or U0126. Data were analysed on the basis of laminae, spinal segment, and AI side. One-way ANOVA with post hoc Tukey’s test, # p
    Figure Legend Snippet: Immunofluorescence analysis indicates the effect of ERK inhibitor on acid injection-induced ERK activation in the spinal dorsal horn. U0126 or vehicle (doses as Fig. 7 ) was injected 5 days after the 2nd AI. ( A ) Immunofluorescence reveals the p-ERK distribution in the L4/5 ipsilateral spinal cords before the 1st AI (Pre) and 3 h post U0126 or DMSO injection. Upper panels: Sham; lower panels: OVX. Scale bar: 100 μm. ( B ) Comparisons of p-ERK-ir cell numbers among the sham and OVX groups with DMSO or U0126. Data were analysed on the basis of laminae, spinal segment, and AI side. One-way ANOVA with post hoc Tukey’s test, # p

    Techniques Used: Immunofluorescence, Injection, Activation Assay

    11) Product Images from "TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts"

    Article Title: TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202323

    Inhibition of ERK pathway modulates UVA-induced MMP-1 and IL-8 production in UVA-irradiated senescent HDFs. (A) Senescent HDFs were treated overnight with different concentrations of U0126 (ERK inhibitor) and irradiated with UVA (1 J/cm 2 ). Cells were cultured for 4 h and protein levels of phosphorylated/total ERK were analyzed by western blotting. Anti-β-tubulin monoclonal antibody was used as a loading control. The images shown are representative of three independent experiments. (B) Total cellular ROS levels were determined by measuring DCF-DA levels using a spectrofluorometer. % ROS production in response to UVA is shown in the graph. Data are presented as percentage compared to the control (DMSO-treated cells). (C) MMP-1 and (D) IL-8 secretion levels in the culture supernatant were measured by ELISA. The values are mean ± SEM of three different experiments. *p
    Figure Legend Snippet: Inhibition of ERK pathway modulates UVA-induced MMP-1 and IL-8 production in UVA-irradiated senescent HDFs. (A) Senescent HDFs were treated overnight with different concentrations of U0126 (ERK inhibitor) and irradiated with UVA (1 J/cm 2 ). Cells were cultured for 4 h and protein levels of phosphorylated/total ERK were analyzed by western blotting. Anti-β-tubulin monoclonal antibody was used as a loading control. The images shown are representative of three independent experiments. (B) Total cellular ROS levels were determined by measuring DCF-DA levels using a spectrofluorometer. % ROS production in response to UVA is shown in the graph. Data are presented as percentage compared to the control (DMSO-treated cells). (C) MMP-1 and (D) IL-8 secretion levels in the culture supernatant were measured by ELISA. The values are mean ± SEM of three different experiments. *p

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

    12) Product Images from "Reactive Oxygen Species-Mediated c-Jun NH2-Terminal Kinase Activation Contributes to Hepatitis B Virus X Protein-Induced Autophagy via Regulation of the Beclin-1/Bcl-2 Interaction"

    Article Title: Reactive Oxygen Species-Mediated c-Jun NH2-Terminal Kinase Activation Contributes to Hepatitis B Virus X Protein-Induced Autophagy via Regulation of the Beclin-1/Bcl-2 Interaction

    Journal: Journal of Virology

    doi: 10.1128/JVI.00001-17

    The JNK signaling pathway is crucial for HBx-induced autophagosome formation. (A) HepG2 cells were transfected with increasing doses of pHBx. At 24 h posttransfection, Western blot analysis was performed with the antibodies indicated. (Left panel) Levels of p-ERK, p-p38, and p-JNK relative to the level of GAPDH were examined by densitometric analysis, and the value from empty-vector-transfected cells was set at 1.0 ( n = 4). (B) HepG2 cells were pretreated with the ERK inhibitor U0126 (10 μM), the p38 inhibitor SB202190 (10 μM), or the JNK inhibitor SP600125 (10 μM) for 30 min and then transfected with pHBx for 48 h. Western blot analysis was performed with the antibodies indicated. (Left panel) Levels of p-ERK, p-p38, and p-JNK relative to the level of GAPDH were examined by densitometric analysis, and the value from empty-vector-transfected cells was set at 1.0 ( n = 5). *, P
    Figure Legend Snippet: The JNK signaling pathway is crucial for HBx-induced autophagosome formation. (A) HepG2 cells were transfected with increasing doses of pHBx. At 24 h posttransfection, Western blot analysis was performed with the antibodies indicated. (Left panel) Levels of p-ERK, p-p38, and p-JNK relative to the level of GAPDH were examined by densitometric analysis, and the value from empty-vector-transfected cells was set at 1.0 ( n = 4). (B) HepG2 cells were pretreated with the ERK inhibitor U0126 (10 μM), the p38 inhibitor SB202190 (10 μM), or the JNK inhibitor SP600125 (10 μM) for 30 min and then transfected with pHBx for 48 h. Western blot analysis was performed with the antibodies indicated. (Left panel) Levels of p-ERK, p-p38, and p-JNK relative to the level of GAPDH were examined by densitometric analysis, and the value from empty-vector-transfected cells was set at 1.0 ( n = 5). *, P

    Techniques Used: Transfection, Western Blot, Plasmid Preparation

    13) Product Images from "Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis"

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis

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

    doi: 10.1073/pnas.1010249107

    ( A ) Cdc42 deficiency leads to ERK activation. Pan T cells were purified from spleen and subjected to anti-phosphorylated ERK (anti-pERK) Western blotting. Total ERK also was blotted as loading control. Data shown are representative of three independent experiments ( Left ). Alternatively, splenocytes were stained with anti-CD4, anti-CD62L, and anti-CD44 antibodies, fixed, permeabilized, and stained with anti-pERK. The naive cells were gated and analyzed for pERK by flow cytometry ( Right ). Data shown are representative of five mice. ( B and C ) ERK activation is required for loss of Cdc42 -induced T-cell hyperproliferation. Naive T cells were purified and cultured with or without the ERK inhibitor U0126 in the presence or absence of 10 μg/mL plate-bound anti-CD3 and 2 μg/mL anti-CD28 antibodies. The cell growth rate ( B ) and IL-2 production ( C ) were assayed. Data are shown as means ± SD; n = 5. ** P
    Figure Legend Snippet: ( A ) Cdc42 deficiency leads to ERK activation. Pan T cells were purified from spleen and subjected to anti-phosphorylated ERK (anti-pERK) Western blotting. Total ERK also was blotted as loading control. Data shown are representative of three independent experiments ( Left ). Alternatively, splenocytes were stained with anti-CD4, anti-CD62L, and anti-CD44 antibodies, fixed, permeabilized, and stained with anti-pERK. The naive cells were gated and analyzed for pERK by flow cytometry ( Right ). Data shown are representative of five mice. ( B and C ) ERK activation is required for loss of Cdc42 -induced T-cell hyperproliferation. Naive T cells were purified and cultured with or without the ERK inhibitor U0126 in the presence or absence of 10 μg/mL plate-bound anti-CD3 and 2 μg/mL anti-CD28 antibodies. The cell growth rate ( B ) and IL-2 production ( C ) were assayed. Data are shown as means ± SD; n = 5. ** P

    Techniques Used: Activation Assay, Purification, Western Blot, Staining, Flow Cytometry, Cytometry, Mouse Assay, Cell Culture

    14) Product Images from "Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function"

    Article Title: Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045276

    T cells from dlg1 knockout mice show normal TCR-induced tyrosine- and alternative p38- phosphorylation. (A) Purified CD4 + T cells from dlg1 flox/flox or dlg1 flox/flox :CD4 cre mice were stimulated with anti-CD3 antibody for the indicated times. Whole cell lysates were immunoblotted with anti-phosphotyrosine antibody. (B) (Top panel) Expanded T cells from dlg1 wt;BG or dlg1 ko;BG mice were restimulated with anti-CD3 and anti-CD28 antibodies for 30 minutes in the absence or presence of an Insolution p38 (InS) or U0126 Erk (E) inhibitor. Whole cell lysates were then immunoblotted with anti-phospho-p38 (T180/Y182) followed by anti-p38 to assess loading. (Bottom panel) Levels of induced p38 phosphorylation relative to corresponding unstimulated samples were determined by densitometry and normalized according to loading controls (n = 3 each for dlg1 wt;BG and dlg1 ko;BG ). Data represent mean +/− StDev. n.s. = not significant, (p = 0.7236).
    Figure Legend Snippet: T cells from dlg1 knockout mice show normal TCR-induced tyrosine- and alternative p38- phosphorylation. (A) Purified CD4 + T cells from dlg1 flox/flox or dlg1 flox/flox :CD4 cre mice were stimulated with anti-CD3 antibody for the indicated times. Whole cell lysates were immunoblotted with anti-phosphotyrosine antibody. (B) (Top panel) Expanded T cells from dlg1 wt;BG or dlg1 ko;BG mice were restimulated with anti-CD3 and anti-CD28 antibodies for 30 minutes in the absence or presence of an Insolution p38 (InS) or U0126 Erk (E) inhibitor. Whole cell lysates were then immunoblotted with anti-phospho-p38 (T180/Y182) followed by anti-p38 to assess loading. (Bottom panel) Levels of induced p38 phosphorylation relative to corresponding unstimulated samples were determined by densitometry and normalized according to loading controls (n = 3 each for dlg1 wt;BG and dlg1 ko;BG ). Data represent mean +/− StDev. n.s. = not significant, (p = 0.7236).

    Techniques Used: Knock-Out, Mouse Assay, Purification

    15) Product Images from "Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation"

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046480

    Cdt2 phosphorylation after UV irradiation is caffeine- and MAP kinase-inhibitor sensitive. A. HeLa cells synchronized in the G1 phase were treated with the indicated inhibitors for 2 h and UV-irradiated (+) or not (−). Cell extract was blotted with Cdt1 and Cdt2 antibodies. Inhibitors used were: ERKi, U0126; JNKi, SP600125; p38i, SB202190. The asterisk indicates a non-specific band, and used as a loading control. B. Asynchronously growing HeLa cells were treated with the indicated MAP kinase inhibitors and UV-irradiated (+) or not. One hour later, cells were examined for Cdt2 phosphorylation levels and Cdt1 protein levels. Cdt2 phosphorylation levels were examined using Phos-tag gel. C. Target specificity of inhibitors. Activation of each MAP kinase was examined using specific phosphopeptide antibodies (Erk1 and 2, JNK or p38) before (−) and after (+) UV-irradiation (C-1 and 2). Cells, treated or not with the indicated inhibitors for 2 h, were UV-irradiated or not. One hour later, cell extracts were prepared and their phosphorylation levels of ERK (Erk1 and Erk2), Jun, and MAPKAPK2 (MK2) were examined(C-2). Note that U0126 inhibits the ERK pathway by inhibiting ERK1-activating kinases MEK1 and MEK2. Jun is a JNK substrate and MAPKAPK2 is a p38 substrate. Arrow in MK2 Western blotting indicates the phosphorylated form of MK2. The asterisk indicates a non-specific band.
    Figure Legend Snippet: Cdt2 phosphorylation after UV irradiation is caffeine- and MAP kinase-inhibitor sensitive. A. HeLa cells synchronized in the G1 phase were treated with the indicated inhibitors for 2 h and UV-irradiated (+) or not (−). Cell extract was blotted with Cdt1 and Cdt2 antibodies. Inhibitors used were: ERKi, U0126; JNKi, SP600125; p38i, SB202190. The asterisk indicates a non-specific band, and used as a loading control. B. Asynchronously growing HeLa cells were treated with the indicated MAP kinase inhibitors and UV-irradiated (+) or not. One hour later, cells were examined for Cdt2 phosphorylation levels and Cdt1 protein levels. Cdt2 phosphorylation levels were examined using Phos-tag gel. C. Target specificity of inhibitors. Activation of each MAP kinase was examined using specific phosphopeptide antibodies (Erk1 and 2, JNK or p38) before (−) and after (+) UV-irradiation (C-1 and 2). Cells, treated or not with the indicated inhibitors for 2 h, were UV-irradiated or not. One hour later, cell extracts were prepared and their phosphorylation levels of ERK (Erk1 and Erk2), Jun, and MAPKAPK2 (MK2) were examined(C-2). Note that U0126 inhibits the ERK pathway by inhibiting ERK1-activating kinases MEK1 and MEK2. Jun is a JNK substrate and MAPKAPK2 is a p38 substrate. Arrow in MK2 Western blotting indicates the phosphorylated form of MK2. The asterisk indicates a non-specific band.

    Techniques Used: Irradiation, Activation Assay, Western Blot

    16) Product Images from "Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice"

    Article Title: Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.078667

    Treatment with a MEK inhibitor attenuates ectopic budding and distal WD expansion in RetY1015F mutant UT explant cultures. ( A , B ) Phase-contrast time-lapse images at indicated timepoints of control (A, Ret EGFP/+ ) and mutant (B, Ret Y1015F/EGFP ) UTs, beginning at E10 with vehicle (top row) or MEK inhibitor U0126 (10 μM, bottom row). In controls, U0126 treatment attenuates budding from distal WD. In mutants, multiple ectopic buds and distal expansion of WDs is inhibited in presence of U0126, indicating increased ERK signaling as a mechanism of these WD defects. Insets are live EGFP images clearly depicting the WD and budding regions. Red arrowheads at 24 hours, mesenchyme; red arrows at 48 hours in insets in B, distal WD/CND. ( C ) Double immunostaining of mutant UT cultures (48 hours) with Pax2 (green) and E-cad (red) show no Pax2 expression (outlined) in the mesenchyme in the mutants treated with U0126 compared with vehicle-treated cultures. This suggests that normal mesonephric domain is established by inhibiting MAPK activity in the mutants. ( D ) Double immunostaining of E11 control UT cultures (48 hours) with Pax2 (green) and E-cad (red) grown in presence of U0126 show common nephric duct (CND) aplasia (red arrowhead). The vehicle-treated CNDs are normally connected to the cloaca.
    Figure Legend Snippet: Treatment with a MEK inhibitor attenuates ectopic budding and distal WD expansion in RetY1015F mutant UT explant cultures. ( A , B ) Phase-contrast time-lapse images at indicated timepoints of control (A, Ret EGFP/+ ) and mutant (B, Ret Y1015F/EGFP ) UTs, beginning at E10 with vehicle (top row) or MEK inhibitor U0126 (10 μM, bottom row). In controls, U0126 treatment attenuates budding from distal WD. In mutants, multiple ectopic buds and distal expansion of WDs is inhibited in presence of U0126, indicating increased ERK signaling as a mechanism of these WD defects. Insets are live EGFP images clearly depicting the WD and budding regions. Red arrowheads at 24 hours, mesenchyme; red arrows at 48 hours in insets in B, distal WD/CND. ( C ) Double immunostaining of mutant UT cultures (48 hours) with Pax2 (green) and E-cad (red) show no Pax2 expression (outlined) in the mesenchyme in the mutants treated with U0126 compared with vehicle-treated cultures. This suggests that normal mesonephric domain is established by inhibiting MAPK activity in the mutants. ( D ) Double immunostaining of E11 control UT cultures (48 hours) with Pax2 (green) and E-cad (red) grown in presence of U0126 show common nephric duct (CND) aplasia (red arrowhead). The vehicle-treated CNDs are normally connected to the cloaca.

    Techniques Used: Mutagenesis, Double Immunostaining, Expressing, Activity Assay

    17) Product Images from "Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages"

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2015.00650

    ERK-dependent production of anti-listerial effector molecules and autophagy protects against Listeria infection of BMDM . WT and Cyld −/− BMDM (1 × 10 6 /well) were cultivated and stimulated with IFN-γ (100 U/ml) for 24 h. Thereafter, the indicated groups of BMDM were infected with Lm (MOI 5:1) or left uninfected (0 h p.i.). (A) IFN-γ-stimulated BMDM were either untreated or treated with the ERK inhibitor U0126 (1 μM) beginning 2 h before infection. Intracellular ROS was determined by flow cytometry using a ROS detection kit at the indicated time points. Data show the mean + SD of triplicate wells per group. (B) Uninfected and infected, IFN-γ-stimulated WT, and Cyld −/− BMDM were treated with the ERK inhibitor U0126 (1 μM) beginning 2 h before infection as indicated. The supernatant was harvested before and 24 h p.i. NO was determined photometrically in the supernatant using a Griess assay kit. Data show the mean + SD of triplicate wells per group. (C) Proteins were isolated from IFN-γ-stimulated, uninfected (0 h p.i.), and infected (2 h p.i.) WT and Cyld −/− BMDM and WB analysis for the autophagosomal marker LC3B-II and GAPDH was performed. LC3B-II expression was determined using a densitometer and data show the mean of three WB performed with independent samples. (D) Immunofluorescence images from uninfected and Lm-infected WT and Cyld −/− BMDM are shown. Cells (5 × 10 5 BMDM per slide) were either untreated or treated with RIPK2 siRNA (D3, beginning 48 h before infection) or the ERK inhibitor U0126 (D4, 1 μM, beginning 2 h before infection). BMDM were stained for Listeria (red), LC3B-II (green), and the nucleus (DAPI, blue). (E) Two hours p.i., the number of LC3B-II puncta per cell from the experimental groups shown in (D) was determined microscopically. Data show the mean + SD of three slides and 100 BMDM/slide per group. (F) CFUs were determined in 1 × 10 6 Lm-infected WT and Cyld −/− BMDM, which were either untreated or treated with ERK inhibitor U0126 (10 μM) beginning 2 h before infection. Data show the mean + SD of triplicate wells per group. In (A–F) , data represent one of the two independent experiments.
    Figure Legend Snippet: ERK-dependent production of anti-listerial effector molecules and autophagy protects against Listeria infection of BMDM . WT and Cyld −/− BMDM (1 × 10 6 /well) were cultivated and stimulated with IFN-γ (100 U/ml) for 24 h. Thereafter, the indicated groups of BMDM were infected with Lm (MOI 5:1) or left uninfected (0 h p.i.). (A) IFN-γ-stimulated BMDM were either untreated or treated with the ERK inhibitor U0126 (1 μM) beginning 2 h before infection. Intracellular ROS was determined by flow cytometry using a ROS detection kit at the indicated time points. Data show the mean + SD of triplicate wells per group. (B) Uninfected and infected, IFN-γ-stimulated WT, and Cyld −/− BMDM were treated with the ERK inhibitor U0126 (1 μM) beginning 2 h before infection as indicated. The supernatant was harvested before and 24 h p.i. NO was determined photometrically in the supernatant using a Griess assay kit. Data show the mean + SD of triplicate wells per group. (C) Proteins were isolated from IFN-γ-stimulated, uninfected (0 h p.i.), and infected (2 h p.i.) WT and Cyld −/− BMDM and WB analysis for the autophagosomal marker LC3B-II and GAPDH was performed. LC3B-II expression was determined using a densitometer and data show the mean of three WB performed with independent samples. (D) Immunofluorescence images from uninfected and Lm-infected WT and Cyld −/− BMDM are shown. Cells (5 × 10 5 BMDM per slide) were either untreated or treated with RIPK2 siRNA (D3, beginning 48 h before infection) or the ERK inhibitor U0126 (D4, 1 μM, beginning 2 h before infection). BMDM were stained for Listeria (red), LC3B-II (green), and the nucleus (DAPI, blue). (E) Two hours p.i., the number of LC3B-II puncta per cell from the experimental groups shown in (D) was determined microscopically. Data show the mean + SD of three slides and 100 BMDM/slide per group. (F) CFUs were determined in 1 × 10 6 Lm-infected WT and Cyld −/− BMDM, which were either untreated or treated with ERK inhibitor U0126 (10 μM) beginning 2 h before infection. Data show the mean + SD of triplicate wells per group. In (A–F) , data represent one of the two independent experiments.

    Techniques Used: Infection, Flow Cytometry, Cytometry, Griess Assay, Isolation, Western Blot, Marker, Expressing, Immunofluorescence, Staining

    18) Product Images from "ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells"

    Article Title: ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0568-4

    Inhibition of ERK phosphorylation and immunophenotyping for EC markers. I Concentration -dependent effect of ERK inhibitor (U0126). Three different concentrations (0.5, 1.0, and 5.0 μM) of U0126 were used. Western blot analysis showed significant inhibition of p-ERK by 1.0 and 5.0 μM of U0126. However, 5.0 μM of U0126 showed the highest inhibition among all three different concentrations. Phospho-ERK was normalized to its total protein expression. II Flow cytometric analysis of PECAM1 (CD31) with ERK inhibitor (U0126). Three different groups treated with 5.0 μM of U0126: AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), and AMSCs with EGM and MMP-14 siRNA ( C ). Flow cytometry data were analyzed to show the significant differences between the groups ( D ). III Flow cytometric analysis of VE-cadherin (CD144) with ERK inhibitor (U0126). Three different groups were treated with 5.0 μM of U0126: AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), and AMSCs with EGM and MMP-14 siRNA ( C ). Flow cytometry data were analyzed to show the significant differences with or without U0126 ( D ). * p
    Figure Legend Snippet: Inhibition of ERK phosphorylation and immunophenotyping for EC markers. I Concentration -dependent effect of ERK inhibitor (U0126). Three different concentrations (0.5, 1.0, and 5.0 μM) of U0126 were used. Western blot analysis showed significant inhibition of p-ERK by 1.0 and 5.0 μM of U0126. However, 5.0 μM of U0126 showed the highest inhibition among all three different concentrations. Phospho-ERK was normalized to its total protein expression. II Flow cytometric analysis of PECAM1 (CD31) with ERK inhibitor (U0126). Three different groups treated with 5.0 μM of U0126: AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), and AMSCs with EGM and MMP-14 siRNA ( C ). Flow cytometry data were analyzed to show the significant differences between the groups ( D ). III Flow cytometric analysis of VE-cadherin (CD144) with ERK inhibitor (U0126). Three different groups were treated with 5.0 μM of U0126: AMSCs with EGM ( A ), AMSCs with EGM and MMP-2 siRNA ( B ), and AMSCs with EGM and MMP-14 siRNA ( C ). Flow cytometry data were analyzed to show the significant differences with or without U0126 ( D ). * p

    Techniques Used: Inhibition, Concentration Assay, Western Blot, Expressing, Flow Cytometry, Cytometry

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    Article Snippet: .. Although results using inhibitors such as U0126 and geldanamycin suggest that ERK1/2 may be upstream of choline kinase, additional information, e.g., the influence of transfection with ERK or dominant negative ERK or MEK on choline metabolism will help strengthen the argument. .. We have chosen to develop an endpoint for drugs, which act on the Raf-1-MEK-ERK signal transduction pathway.

    Transfection:

    Article Title: Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin
    Article Snippet: .. Although results using inhibitors such as U0126 and geldanamycin suggest that ERK1/2 may be upstream of choline kinase, additional information, e.g., the influence of transfection with ERK or dominant negative ERK or MEK on choline metabolism will help strengthen the argument. .. We have chosen to develop an endpoint for drugs, which act on the Raf-1-MEK-ERK signal transduction pathway.

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    Article Title: Mitogen-activated Protein Kinase (MAPK)-regulated Interactions between Osterix and Runx2 Are Critical for the Transcriptional Osteogenic Program *
    Article Snippet: We further characterized the interaction between endogenous Runx2 and Osx in primary cultures of mouse calvarial osteoblasts treated with SB203580, U0126, or both inhibitors.

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    Article Snippet: The reagents used in this study were obtained from the following sources: tissue culture medium and fetal bovine serum from Invitrogen; U0126 from Calbiochem; mouse anti-Runx2 antibody from MBL; phosphoserine antibody from ABCam; and M2 and M2 horseradish peroxidase-conjugated antibody from Sigma.

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    Millipore erk 1 2 inhibitor u0126
    The anti-apoptotic effect of IGF-1 is abrogated by the inhibitors of the IGF-1 receptor (AG1024), Akt (LY294002), and <t>Erk-1/2</t> <t>(U0126).</t> Retinal ganglion cells (RGCs) were cultured in the absence or presence of U0126 (U, 5 µM), LY294002 (LY, 20 µM), and AG1024 (AG, 10 µM) with or without IGF-1, 200 ng/ml, under normoxia or hypoxia for 24 h. Western blot analysis was performed to detect ( A ) activation of total Akt, and phospho-Akt and phosphorylation of Bad at serine 136, ( B ) activation of total Erk-1/2 and phospho-Erk-1/2, and phosphorylation of Bad at serine 112, and ( C ) the levels of the active capase-3 subunit. β-actin immunoreactivity from the same gel is shown as the internal control. Histograms show the results of the relative protein level normalized to β-actin. D : Apoptotic cells were detected TUNEL analyses. Representative results of three independent experiments are shown. The results are expressed as the means±SEM. *p
    Erk 1 2 Inhibitor U0126, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The anti-apoptotic effect of IGF-1 is abrogated by the inhibitors of the IGF-1 receptor (AG1024), Akt (LY294002), and Erk-1/2 (U0126). Retinal ganglion cells (RGCs) were cultured in the absence or presence of U0126 (U, 5 µM), LY294002 (LY, 20 µM), and AG1024 (AG, 10 µM) with or without IGF-1, 200 ng/ml, under normoxia or hypoxia for 24 h. Western blot analysis was performed to detect ( A ) activation of total Akt, and phospho-Akt and phosphorylation of Bad at serine 136, ( B ) activation of total Erk-1/2 and phospho-Erk-1/2, and phosphorylation of Bad at serine 112, and ( C ) the levels of the active capase-3 subunit. β-actin immunoreactivity from the same gel is shown as the internal control. Histograms show the results of the relative protein level normalized to β-actin. D : Apoptotic cells were detected TUNEL analyses. Representative results of three independent experiments are shown. The results are expressed as the means±SEM. *p

    Journal: Molecular Vision

    Article Title: IGF-1 protects retinal ganglion cells from hypoxia-induced apoptosis by activating the Erk-1/2 and Akt pathways

    doi:

    Figure Lengend Snippet: The anti-apoptotic effect of IGF-1 is abrogated by the inhibitors of the IGF-1 receptor (AG1024), Akt (LY294002), and Erk-1/2 (U0126). Retinal ganglion cells (RGCs) were cultured in the absence or presence of U0126 (U, 5 µM), LY294002 (LY, 20 µM), and AG1024 (AG, 10 µM) with or without IGF-1, 200 ng/ml, under normoxia or hypoxia for 24 h. Western blot analysis was performed to detect ( A ) activation of total Akt, and phospho-Akt and phosphorylation of Bad at serine 136, ( B ) activation of total Erk-1/2 and phospho-Erk-1/2, and phosphorylation of Bad at serine 112, and ( C ) the levels of the active capase-3 subunit. β-actin immunoreactivity from the same gel is shown as the internal control. Histograms show the results of the relative protein level normalized to β-actin. D : Apoptotic cells were detected TUNEL analyses. Representative results of three independent experiments are shown. The results are expressed as the means±SEM. *p

    Article Snippet: The IGF-1 reporter inhibitor AG1024 (121,767) and the Erk-1/2 inhibitor U0126 (662,005) were purchased from Calbiochem (La Jolla, CA).

    Techniques: Cell Culture, Western Blot, Activation Assay, TUNEL Assay

    Effect of MEK and MAPK inhibitors on claudin-5 expression. Cells were treated with medium alone, 10 µM U0126, 10 µM SP600125, or 20 µM SB208580 for 48 hours. (A) Whole cell lysates were analyzed for claudin-5, VE-cadherin, and the phosphorylated and total forms of ERK 1/2, HSP27, and c-Jun by Western blot. Tubulin served as the loading control. Representative immunoblots of three separate experiments are shown. (B) Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. *, p

    Journal: PLoS ONE

    Article Title: Anthrax Lethal Toxin Downregulates Claudin-5 Expression in Human Endothelial Tight Junctions

    doi: 10.1371/journal.pone.0062576

    Figure Lengend Snippet: Effect of MEK and MAPK inhibitors on claudin-5 expression. Cells were treated with medium alone, 10 µM U0126, 10 µM SP600125, or 20 µM SB208580 for 48 hours. (A) Whole cell lysates were analyzed for claudin-5, VE-cadherin, and the phosphorylated and total forms of ERK 1/2, HSP27, and c-Jun by Western blot. Tubulin served as the loading control. Representative immunoblots of three separate experiments are shown. (B) Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. *, p

    Article Snippet: U0126 (an inhibitor of MEK-1/2), SB230580 (an inhibitor of p38 MAPK), SP600125 (an inhibitor of JNK), MG132, marimastat, E-64, pepstatin A, Y27632, and ML-7 were purchased from EMD Chemicals (Gibbstown, NJ).

    Techniques: Expressing, Western Blot

    Bidirectional transitions shape SAMHD 1‐mediated restriction of HIV ‐1 MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1 G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1. MDM were co‐infected with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing vpx (VLP‐vpx). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods ; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m. Proposed signalling pathway leading to SAMHD1 phosphorylation in stimulated MDM. Unstimulated (G) and stimulated (H) MDM were treated with inhibitors of RAF (2 μM), B‐RAF (3 μM), MEK1/2 (AS‐703026, 1 μM), JAK 1–3 (1 μM), GSK3 (2 μM), PIM 1–3 (3 μM) and CDK4/6 (1 μM) for 18 h before infection and infected with VSV‐G HIV‐1 GFP. The percentage of infected cells was quantified by FACS 48 h post‐infection. Graphs are representative example of n ≥ 3, mean ± s.e.m., *P‐ value ≤ 0.05; **P‐ value ≤ 0.01; calculated from triplicates, unpaired t ‐test). MDM were treated with a MEK/ERK inhibitor (U0126, 10 μM) for 18 h before infection and where indicated VLP‐vpx was added at the time of infection. Percentage of infected cells were detected by FACS 48 h post‐infection. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins ( n = 3, mean ± s.e.m.; (ns) non‐significant; ***P‐ value ≤ 0.001, unpaired t ‐test). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages

    doi: 10.15252/embj.201696025

    Figure Lengend Snippet: Bidirectional transitions shape SAMHD 1‐mediated restriction of HIV ‐1 MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1 G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1. MDM were co‐infected with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing vpx (VLP‐vpx). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods ; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m. Proposed signalling pathway leading to SAMHD1 phosphorylation in stimulated MDM. Unstimulated (G) and stimulated (H) MDM were treated with inhibitors of RAF (2 μM), B‐RAF (3 μM), MEK1/2 (AS‐703026, 1 μM), JAK 1–3 (1 μM), GSK3 (2 μM), PIM 1–3 (3 μM) and CDK4/6 (1 μM) for 18 h before infection and infected with VSV‐G HIV‐1 GFP. The percentage of infected cells was quantified by FACS 48 h post‐infection. Graphs are representative example of n ≥ 3, mean ± s.e.m., *P‐ value ≤ 0.05; **P‐ value ≤ 0.01; calculated from triplicates, unpaired t ‐test). MDM were treated with a MEK/ERK inhibitor (U0126, 10 μM) for 18 h before infection and where indicated VLP‐vpx was added at the time of infection. Percentage of infected cells were detected by FACS 48 h post‐infection. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins ( n = 3, mean ± s.e.m.; (ns) non‐significant; ***P‐ value ≤ 0.001, unpaired t ‐test). Source data are available online for this figure.

    Article Snippet: Kinase inhibitors used: CDK4/6 inhibitor (PD 0332991, Palbociclib) from Sigma; MEK/ERK inhibitor U0126 from Calbiochem (San Diego, USA); JAK 1–3 (ruxolitinib), PIM 1–3 (AZD1897), GSK3 (CT99021), and MEK1/2 (AS‐703026), RAF (TAK‐632), B‐RAF (PXL4032) from Selleckchem (Houston, TX, USA), SAHA (vorinostat) from Sigma and panobinostat from Cayman Chemicals (Ann Arbor, MI, USA).

    Techniques: Infection, FACS, Transfection, Cell Culture

    Direct inhibition of ADAM17 more effectively sustained GPIbα retention by cultured iPSC‐derived platelets than suppression of potential upstream activators of ADAM17. Percentages of the GPIbα + population in CD41a + iPSC‐derived platelets treated with the indicated inhibitors during the platelet production phase (days 21–23 or 24). KP‐457 (15 μmol/l), BIRB796 (10 μmol/l), MAPK/extracellular signal‐regulated kinase kinase inhibitor U0126 (10 μmol/l), I k B kinase β inhibitor BMS345541 (10 μmol/l), caspase inhibitor Z‐VAD (10 μmol/l) and protein kinase C inhibitor Ro31‐8220 (3 μmol/l) were used. Inhibition of potential ADAM17 activator molecules failed to prevent GPIbα shedding evoked by cultivation at 37°C. ∗, p

    Journal: Stem Cells Translational Medicine

    Article Title: Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell‐Derived Platelets

    doi: 10.5966/sctm.2016-0104

    Figure Lengend Snippet: Direct inhibition of ADAM17 more effectively sustained GPIbα retention by cultured iPSC‐derived platelets than suppression of potential upstream activators of ADAM17. Percentages of the GPIbα + population in CD41a + iPSC‐derived platelets treated with the indicated inhibitors during the platelet production phase (days 21–23 or 24). KP‐457 (15 μmol/l), BIRB796 (10 μmol/l), MAPK/extracellular signal‐regulated kinase kinase inhibitor U0126 (10 μmol/l), I k B kinase β inhibitor BMS345541 (10 μmol/l), caspase inhibitor Z‐VAD (10 μmol/l) and protein kinase C inhibitor Ro31‐8220 (3 μmol/l) were used. Inhibition of potential ADAM17 activator molecules failed to prevent GPIbα shedding evoked by cultivation at 37°C. ∗, p

    Article Snippet: The panmetalloproteinase inhibitor GM‐6001, p38 MAPK inhibitor SB203580, MAPK/extracellular signal‐regulated kinase kinase (MEK) inhibitor U0126, IκB kinase β (IKKβ) inhibitor BMS345541, caspase inhibitor Z‐VAD, and protein kinase C (PKC) inhibitor Ro31‐8220 were from EMD Millipore (Billerica, MA, http://www.emdmillipore.com ).

    Techniques: Inhibition, Cell Culture, Derivative Assay