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: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/erk inhibitor u0126/product/Millipore
    Average 98 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    erk inhibitor u0126 - by Bioz Stars, 2020-02
    98/100 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 "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

    3) 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

    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 "[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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    Related Articles

    Centrifugation:

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages
    Article Snippet: Cultures with an optical density of 0.1, which corresponds to a dose of 1 × 108 Lm/ml according to a previously established standard growth curve, were pelleted by centrifugation (870 × g , 4°C, 10 min) and the multiplicity of infection MOI was adjusted to 5:1 in DMEM supplemented with 10% FCS, 1% non-essential amino acids, 1% glutamine, and 50 μM 2-mercaptoethanol. .. For the inhibition of ERK, BMDM were treated with the ERK inhibitor U0126 (1 μM; Calbiochem) beginning 2 h before infection according to the manufacturer’s recommendation.

    Stable Transfection:

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation
    Article Snippet: Cell culture HeLa, HEK293 and HEK293 cells stably expressing Cdt2-FLAG, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% CO2 . .. MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126 (Calbiochem) at 10 µM, JNK inhibitor II (SP600125, Calbiochem) at 30 µM and p38 inhibitor SB202190 (Calbiochem) at 20 µM.

    Synthesized:

    Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway
    Article Snippet: The ERK inhibitor U0126 was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). .. The reverse transcription-quantitative polymerase chain reaction primers for CCR7, ANO6, ERK1/2 and GAPDH were synthesized commercially by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

    Blocking Assay:

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation
    Article Snippet: To synchronize cells in the G1 phase, cells blocked in the early S phase using the thymidine and aphidicolin block method were released for 16 h or nocodazole-arrested cells were released for 3 h. Caffeine was used at 10 mM. .. MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126 (Calbiochem) at 10 µM, JNK inhibitor II (SP600125, Calbiochem) at 30 µM and p38 inhibitor SB202190 (Calbiochem) at 20 µM.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: .. Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added. .. Cells were incubated for another 48 h and then analyzed for cell growth rate by a Non-Radioactive Cell Proliferation Assay kit (Promega).

    Incubation:

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages
    Article Snippet: For inhibition of NF-κB, BMDM were treated with IKK inhibitor VII (1 μM; Calbiochem, Darmstadt, Germany) beginning 24 h before infection (concentration and incubation time were pretested before usage). .. For the inhibition of ERK, BMDM were treated with the ERK inhibitor U0126 (1 μM; Calbiochem) beginning 2 h before infection according to the manufacturer’s recommendation.

    Article Title: Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung
    Article Snippet: Apical fluid was replaced with either the JNK inhibitor SP600125 (20 µM) or the ERK inhibitor U0126 (10 µM) (Sigma, Saint Louis, MO). .. Cells on silastic membranes were incubated with the inhibitor for 60 minutes, and then lysed for analysis of signaling activation and TJ protein concentration as described above (N = 9 isolations per group, 1–2 wells per isolation).

    Article Title: ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells
    Article Snippet: ERK inhibition The phosphorylation of ERK1/2 was inhibited by ERK inhibitor U0126 (662005; Calbiochem, USA) to analyze the role of the ERK pathway in the differentiation of AMSCs to ECs before and after MMP-2 or MMP-14 silencing. .. To examine the phosphorylation status of ERK, differentiated AMSCs were incubated in EGM and ERK inhibitor for 3 hours, and tested for ERK activity by western blot analysis.

    Article Title: TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts
    Article Snippet: Prior to UV irradiation, cells were pre-treated with various concentrations of the ERK inhibitor U0126 (Sigma-Aldrich, MO, USA) or TLR4 inhibitor TAK242 (Cayman Chemicals, MI, USA). .. After UVA exposure, the cells were cultured in fresh media and incubated for an additional amount of time before being collected for further analysis.

    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
    Article Snippet: After pretreatment with the ERK inhibitor U0126 (Calbiochem, Billerica, MA, USA) for 1 h and treatment with/without [6]-gingerol for 24 h, cells were harvested, and then protein was extracted with protein lysis buffer (25 mM Tris-Cl, pH 7.5, 250 mM NaCl, 5 mM ethylendiaminetetracetic acid, 1% Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails; Thermo Scientific, Waltham, MA, USA). .. After overnight incubation at 4°C, membranes were washed in TBST and incubated with the appropriate peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added. .. Cells were incubated for another 48 h and then analyzed for cell growth rate by a Non-Radioactive Cell Proliferation Assay kit (Promega).

    Article Title: Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor
    Article Snippet: .. For pathway analysis, 25 μM of the ERK inhibitor U0126 was incubated with NK cells for 30 min prior to the preparation of lysates (Sigma Aldrich). .. NK cells activated through their FcR by immobilized IgG were subject to flow cytometry to determine cell surface expression of IL-21R.

    Activity Assay:

    Article Title: ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells
    Article Snippet: ERK inhibition The phosphorylation of ERK1/2 was inhibited by ERK inhibitor U0126 (662005; Calbiochem, USA) to analyze the role of the ERK pathway in the differentiation of AMSCs to ECs before and after MMP-2 or MMP-14 silencing. .. To examine the phosphorylation status of ERK, differentiated AMSCs were incubated in EGM and ERK inhibitor for 3 hours, and tested for ERK activity by western blot analysis.

    Article Title: 4-1BB Controls Regulatory Activity in Dendritic Cells through Promoting Optimal Expression of Retinal Dehydrogenase
    Article Snippet: For blockade of signaling, the following were used; ERK inhibitor U0126 (5 μM); PI3K inhibitor (5 μM); NFκB inhibitor Bay 11–7082 (1 μM, all from Sigma-Aldrich); Wnt/β-catenin inhibitor XAV939 (5 μM, sc-296704, Santa Cruz Biotechnology). .. The activity of RALDH was determined by ALDEFLUOR staining as described ( ).

    Cell Culture:

    Article Title: Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice
    Article Snippet: .. E10 half GU cultures (each half cultured separately) for determining role of ERK signaling were grown in presence of the ERK inhibitor U0126 (10 μM, Sigma) or with DMSO (vehicle). ..

    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
    Article Snippet: Paragraph title: Cell culture and reagents ... Propofol (Sigma, St. Louis, MO, USA), calcium chelator BAPTA-AM (Sigma, St. Louis, MO, USA), CAMKIIα inhibitor KN93 (Sigma, St. Louis, MO, USA), ERK inhibitor U0126 and JAK1 inhibitor INCB039110 were dissolved in DMSO (Sigma, St. Louis, MO, USA).

    Article Title: 4-1BB Controls Regulatory Activity in Dendritic Cells through Promoting Optimal Expression of Retinal Dehydrogenase
    Article Snippet: 2 ~ 3 × 105 SDC were cultured with a cytokine, GM-CSF (10~1000 pg/ml), or TLR2 ligands Zymosan (0.25 ~ 25 μg/ml, Sigma-Aldrich) and Pam-3-cys (0.1 ~ 10 μg/ml, Invivogen) for 48 h. Where indicated, rat IgG (KLH/G1-2-2, 25 μg/ml), anti-4-1BB (3H3, 25 μg/ml), and inhibitors of signaling pathways, were added at the start of culture. .. For blockade of signaling, the following were used; ERK inhibitor U0126 (5 μM); PI3K inhibitor (5 μM); NFκB inhibitor Bay 11–7082 (1 μM, all from Sigma-Aldrich); Wnt/β-catenin inhibitor XAV939 (5 μM, sc-296704, Santa Cruz Biotechnology).

    Article Title: TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts
    Article Snippet: Prior to UV irradiation, cells were pre-treated with various concentrations of the ERK inhibitor U0126 (Sigma-Aldrich, MO, USA) or TLR4 inhibitor TAK242 (Cayman Chemicals, MI, USA). .. After UVA exposure, the cells were cultured in fresh media and incubated for an additional amount of time before being collected for further analysis.

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: .. Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added. .. Cells were incubated for another 48 h and then analyzed for cell growth rate by a Non-Radioactive Cell Proliferation Assay kit (Promega).

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation
    Article Snippet: Paragraph title: Cell culture ... MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126 (Calbiochem) at 10 µM, JNK inhibitor II (SP600125, Calbiochem) at 30 µM and p38 inhibitor SB202190 (Calbiochem) at 20 µM.

    Expressing:

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation
    Article Snippet: Cell culture HeLa, HEK293 and HEK293 cells stably expressing Cdt2-FLAG, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% CO2 . .. MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126 (Calbiochem) at 10 µM, JNK inhibitor II (SP600125, Calbiochem) at 30 µM and p38 inhibitor SB202190 (Calbiochem) at 20 µM.

    Article Title: Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor
    Article Snippet: Lysates were prepared from human NK cells as previously described following FcR-stimulation by immobilized IgG and assayed for the expression of IL-21R (MAB991; R & D Systems Inc., Minneapolis, MN) or β-Actin as a loading control (clone AC-74; Sigma Aldrich, St. Louis, MO). .. For pathway analysis, 25 μM of the ERK inhibitor U0126 was incubated with NK cells for 30 min prior to the preparation of lysates (Sigma Aldrich).

    Modification:

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation
    Article Snippet: Cell culture HeLa, HEK293 and HEK293 cells stably expressing Cdt2-FLAG, and HEK293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% CO2 . .. MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126 (Calbiochem) at 10 µM, JNK inhibitor II (SP600125, Calbiochem) at 30 µM and p38 inhibitor SB202190 (Calbiochem) at 20 µM.

    Western Blot:

    Article Title: Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function
    Article Snippet: Alternative p38 phosphorylation 3×106 expanded T cells from dlg1wt;BG or dlgko;BG mice were rested for 4 hrs at 37°C, and subsequently pretreated with either 10 uM of “InSolution” p38 inhibitor (506148; Calbiochem), 10 uM of ERK inhibitor U0126 (662005; Calbiochem), or DMSO (control) in the presence of complete RPMI media for 30′ minutes at 37°C. .. Immunoblots were performed with anti-phospho-p38 (Thr180/Tyr182) (3D7) rabbit monoclonal antibody (9215;Cell Signaling), after which the blots were stripped and reprobed with anti-p38α α (C20) rabbit polyclonal antibody (sc-535;Santa Cruz Biotechnologies) to assess loading.

    Article Title: ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells
    Article Snippet: ERK inhibition The phosphorylation of ERK1/2 was inhibited by ERK inhibitor U0126 (662005; Calbiochem, USA) to analyze the role of the ERK pathway in the differentiation of AMSCs to ECs before and after MMP-2 or MMP-14 silencing. .. To examine the phosphorylation status of ERK, differentiated AMSCs were incubated in EGM and ERK inhibitor for 3 hours, and tested for ERK activity by western blot analysis.

    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
    Article Snippet: Paragraph title: 2.8. Western Blot Analysis ... After pretreatment with the ERK inhibitor U0126 (Calbiochem, Billerica, MA, USA) for 1 h and treatment with/without [6]-gingerol for 24 h, cells were harvested, and then protein was extracted with protein lysis buffer (25 mM Tris-Cl, pH 7.5, 250 mM NaCl, 5 mM ethylendiaminetetracetic acid, 1% Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails; Thermo Scientific, Waltham, MA, USA).

    Tail Flick Test:

    Article Title: Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain
    Article Snippet: Intrathecal injection of the ERK MAPK inhibitor in female rats To investigate the involvement of ERK activation in nociceptive sensitization, i.t. injection of the ERK inhibitor U0126 (Sigma-Aldrich, Saint Louis, MO) was performed in female rats. .. Correct needle placement inside the spinal thecal canal was confirmed by the appearance of a brief rapid tail flick.

    Activation Assay:

    Article Title: Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung
    Article Snippet: Apical fluid was replaced with either the JNK inhibitor SP600125 (20 µM) or the ERK inhibitor U0126 (10 µM) (Sigma, Saint Louis, MO). .. Cells on silastic membranes were incubated with the inhibitor for 60 minutes, and then lysed for analysis of signaling activation and TJ protein concentration as described above (N = 9 isolations per group, 1–2 wells per isolation).

    Article Title: Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain
    Article Snippet: .. Intrathecal injection of the ERK MAPK inhibitor in female rats To investigate the involvement of ERK activation in nociceptive sensitization, i.t. injection of the ERK inhibitor U0126 (Sigma-Aldrich, Saint Louis, MO) was performed in female rats. ..

    Protease Inhibitor:

    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
    Article Snippet: .. After pretreatment with the ERK inhibitor U0126 (Calbiochem, Billerica, MA, USA) for 1 h and treatment with/without [6]-gingerol for 24 h, cells were harvested, and then protein was extracted with protein lysis buffer (25 mM Tris-Cl, pH 7.5, 250 mM NaCl, 5 mM ethylendiaminetetracetic acid, 1% Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails; Thermo Scientific, Waltham, MA, USA). .. Quantification of protein concentration was carried out using the Bradford method (Bio-Rad protein assay reagent), and total protein was resuspended in Laemmli sample buffer containing 5% β -mercaptoethanol and heated at 65°C for 10 min. Aliquots containing ~20–50 μ g of total cell proteins were resolved on 8–12% sodium dodecyl sulfate (SDS)-PAGE and then transferred onto nitrocellulose membranes (Amersham, Arlington Heights, IL, USA).

    Infection:

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages
    Article Snippet: .. For the inhibition of ERK, BMDM were treated with the ERK inhibitor U0126 (1 μM; Calbiochem) beginning 2 h before infection according to the manufacturer’s recommendation. ..

    Inhibition:

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages
    Article Snippet: .. For the inhibition of ERK, BMDM were treated with the ERK inhibitor U0126 (1 μM; Calbiochem) beginning 2 h before infection according to the manufacturer’s recommendation. ..

    Article Title: Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung
    Article Snippet: Paragraph title: Inhibition of MAPk Signaling Pathways ... Apical fluid was replaced with either the JNK inhibitor SP600125 (20 µM) or the ERK inhibitor U0126 (10 µM) (Sigma, Saint Louis, MO).

    Article Title: ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells
    Article Snippet: .. ERK inhibition The phosphorylation of ERK1/2 was inhibited by ERK inhibitor U0126 (662005; Calbiochem, USA) to analyze the role of the ERK pathway in the differentiation of AMSCs to ECs before and after MMP-2 or MMP-14 silencing. ..

    Protein Concentration:

    Article Title: Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung
    Article Snippet: Apical fluid was replaced with either the JNK inhibitor SP600125 (20 µM) or the ERK inhibitor U0126 (10 µM) (Sigma, Saint Louis, MO). .. Cells on silastic membranes were incubated with the inhibitor for 60 minutes, and then lysed for analysis of signaling activation and TJ protein concentration as described above (N = 9 isolations per group, 1–2 wells per isolation).

    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
    Article Snippet: After pretreatment with the ERK inhibitor U0126 (Calbiochem, Billerica, MA, USA) for 1 h and treatment with/without [6]-gingerol for 24 h, cells were harvested, and then protein was extracted with protein lysis buffer (25 mM Tris-Cl, pH 7.5, 250 mM NaCl, 5 mM ethylendiaminetetracetic acid, 1% Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails; Thermo Scientific, Waltham, MA, USA). .. Quantification of protein concentration was carried out using the Bradford method (Bio-Rad protein assay reagent), and total protein was resuspended in Laemmli sample buffer containing 5% β -mercaptoethanol and heated at 65°C for 10 min. Aliquots containing ~20–50 μ g of total cell proteins were resolved on 8–12% sodium dodecyl sulfate (SDS)-PAGE and then transferred onto nitrocellulose membranes (Amersham, Arlington Heights, IL, USA).

    Polymerase Chain Reaction:

    Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway
    Article Snippet: The ERK inhibitor U0126 was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). .. The reverse transcription-quantitative polymerase chain reaction primers for CCR7, ANO6, ERK1/2 and GAPDH were synthesized commercially by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

    Injection:

    Article Title: Ovarian Hormone-dependent and Spinal ERK Activation-regulated Nociceptive Hypersensitivity in Female Rats with Acid Injection-induced Chronic Widespread Muscle Pain
    Article Snippet: .. Intrathecal injection of the ERK MAPK inhibitor in female rats To investigate the involvement of ERK activation in nociceptive sensitization, i.t. injection of the ERK inhibitor U0126 (Sigma-Aldrich, Saint Louis, MO) was performed in female rats. ..

    Recombinant:

    Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway
    Article Snippet: Recombinant human CCL21 was obtained from Cyagen Biosciences, Inc. (Santa Clara, CA, USA). .. The ERK inhibitor U0126 was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

    Organ Culture:

    Article Title: Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice
    Article Snippet: Paragraph title: Organ culture ... E10 half GU cultures (each half cultured separately) for determining role of ERK signaling were grown in presence of the ERK inhibitor U0126 (10 μM, Sigma) or with DMSO (vehicle).

    Mutagenesis:

    Article Title: Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice
    Article Snippet: Whole-mount cultures of UT or metanephros alone from mutant and control embryos were grown in serum-free defined media on transwell filters (0.4 μm) in six-well plates ( ). .. E10 half GU cultures (each half cultured separately) for determining role of ERK signaling were grown in presence of the ERK inhibitor U0126 (10 μM, Sigma) or with DMSO (vehicle).

    Isolation:

    Article Title: Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung
    Article Snippet: Apical fluid was replaced with either the JNK inhibitor SP600125 (20 µM) or the ERK inhibitor U0126 (10 µM) (Sigma, Saint Louis, MO). .. Cells on silastic membranes were incubated with the inhibitor for 60 minutes, and then lysed for analysis of signaling activation and TJ protein concentration as described above (N = 9 isolations per group, 1–2 wells per isolation).

    Mouse Assay:

    Article Title: Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function
    Article Snippet: .. Alternative p38 phosphorylation 3×106 expanded T cells from dlg1wt;BG or dlgko;BG mice were rested for 4 hrs at 37°C, and subsequently pretreated with either 10 uM of “InSolution” p38 inhibitor (506148; Calbiochem), 10 uM of ERK inhibitor U0126 (662005; Calbiochem), or DMSO (control) in the presence of complete RPMI media for 30′ minutes at 37°C. .. Cells were then restimulated with platebound antibody (5 ug anti-CD3 and 20 ug anti-CD28) for 30 minutes at 37°C followed by lysis in TNE buffer (50 mM Tris, 1% Nonidet P-40, 2 mM EDTA, pH 8.0) plus protease (leupeptin and aprotinin 10 ug/ml each, 1 mM PMSF) and phosphatase (1 mM NaVO4 ) inhibitors for 20 minutes on ice.

    Staining:

    Article Title: 4-1BB Controls Regulatory Activity in Dendritic Cells through Promoting Optimal Expression of Retinal Dehydrogenase
    Article Snippet: For blockade of signaling, the following were used; ERK inhibitor U0126 (5 μM); PI3K inhibitor (5 μM); NFκB inhibitor Bay 11–7082 (1 μM, all from Sigma-Aldrich); Wnt/β-catenin inhibitor XAV939 (5 μM, sc-296704, Santa Cruz Biotechnology). .. The activity of RALDH was determined by ALDEFLUOR staining as described ( ).

    Purification:

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: Naive T cells from spleen were purified by FACS of TCRβ+ /CD62Lhigh /CD44low cells. .. Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added.

    SDS Page:

    Article Title: Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function
    Article Snippet: Alternative p38 phosphorylation 3×106 expanded T cells from dlg1wt;BG or dlgko;BG mice were rested for 4 hrs at 37°C, and subsequently pretreated with either 10 uM of “InSolution” p38 inhibitor (506148; Calbiochem), 10 uM of ERK inhibitor U0126 (662005; Calbiochem), or DMSO (control) in the presence of complete RPMI media for 30′ minutes at 37°C. .. Cellular debris was spun down at 12,000 RPM for 20 minutes at 4°C and cleared lysates were used to assess protein phosphorylation by separation on a 10% SDS-PAGE.

    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
    Article Snippet: After pretreatment with the ERK inhibitor U0126 (Calbiochem, Billerica, MA, USA) for 1 h and treatment with/without [6]-gingerol for 24 h, cells were harvested, and then protein was extracted with protein lysis buffer (25 mM Tris-Cl, pH 7.5, 250 mM NaCl, 5 mM ethylendiaminetetracetic acid, 1% Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails; Thermo Scientific, Waltham, MA, USA). .. Quantification of protein concentration was carried out using the Bradford method (Bio-Rad protein assay reagent), and total protein was resuspended in Laemmli sample buffer containing 5% β -mercaptoethanol and heated at 65°C for 10 min. Aliquots containing ~20–50 μ g of total cell proteins were resolved on 8–12% sodium dodecyl sulfate (SDS)-PAGE and then transferred onto nitrocellulose membranes (Amersham, Arlington Heights, IL, USA).

    Article Title: Activation of the FcgammaReceptorIIIa on human natural killer cells leads to increased expression of functional interleukin-21 receptor
    Article Snippet: Lysates were separated on a 10% SDS-PAGE gels, transferred to a nitrocellulose membrane, and blocked in 5% nonfat milk prior to incubation with primary and secondary antibodies. .. For pathway analysis, 25 μM of the ERK inhibitor U0126 was incubated with NK cells for 30 min prior to the preparation of lysates (Sigma Aldrich).

    Proliferation Assay:

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added. .. Cells were incubated for another 48 h and then analyzed for cell growth rate by a Non-Radioactive Cell Proliferation Assay kit (Promega).

    Irradiation:

    Article Title: TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts
    Article Snippet: .. Prior to UV irradiation, cells were pre-treated with various concentrations of the ERK inhibitor U0126 (Sigma-Aldrich, MO, USA) or TLR4 inhibitor TAK242 (Cayman Chemicals, MI, USA). .. Immediately before irradiation, the culture medium was removed and cells were washed twice in PBS.

    Article Title: TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts
    Article Snippet: .. Drug treatment Prior to UV irradiation, cells were pre-treated with various concentrations of the ERK inhibitor U0126 (Sigma-Aldrich, MO, USA) or TLR4 inhibitor TAK242 (Cayman Chemicals, MI, USA). .. Immediately before irradiation, the culture medium was removed and cells were washed twice in PBS.

    In Vitro:

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages
    Article Snippet: Paragraph title: In vitro Infection of BMDM with Lm ... For the inhibition of ERK, BMDM were treated with the ERK inhibitor U0126 (1 μM; Calbiochem) beginning 2 h before infection according to the manufacturer’s recommendation.

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: Paragraph title: In Vitro Proliferation and IL-2 Production. ... Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added.

    Concentration Assay:

    Article Title: Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages
    Article Snippet: For inhibition of NF-κB, BMDM were treated with IKK inhibitor VII (1 μM; Calbiochem, Darmstadt, Germany) beginning 24 h before infection (concentration and incubation time were pretested before usage). .. For the inhibition of ERK, BMDM were treated with the ERK inhibitor U0126 (1 μM; Calbiochem) beginning 2 h before infection according to the manufacturer’s recommendation.

    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
    Article Snippet: Propofol (Sigma, St. Louis, MO, USA), calcium chelator BAPTA-AM (Sigma, St. Louis, MO, USA), CAMKIIα inhibitor KN93 (Sigma, St. Louis, MO, USA), ERK inhibitor U0126 and JAK1 inhibitor INCB039110 were dissolved in DMSO (Sigma, St. Louis, MO, USA). .. The final concentration of DMSO was adjusted to 0.01% for each solution to avoid possible nonspecific effects.

    Article Title: ERK signaling is required for VEGF-A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells
    Article Snippet: ERK inhibition The phosphorylation of ERK1/2 was inhibited by ERK inhibitor U0126 (662005; Calbiochem, USA) to analyze the role of the ERK pathway in the differentiation of AMSCs to ECs before and after MMP-2 or MMP-14 silencing. .. ERK inhibitor was applied to the AMSCs in EGM at a final concentration of 5 μM.

    Lysis:

    Article Title: Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function
    Article Snippet: Alternative p38 phosphorylation 3×106 expanded T cells from dlg1wt;BG or dlgko;BG mice were rested for 4 hrs at 37°C, and subsequently pretreated with either 10 uM of “InSolution” p38 inhibitor (506148; Calbiochem), 10 uM of ERK inhibitor U0126 (662005; Calbiochem), or DMSO (control) in the presence of complete RPMI media for 30′ minutes at 37°C. .. Cells were then restimulated with platebound antibody (5 ug anti-CD3 and 20 ug anti-CD28) for 30 minutes at 37°C followed by lysis in TNE buffer (50 mM Tris, 1% Nonidet P-40, 2 mM EDTA, pH 8.0) plus protease (leupeptin and aprotinin 10 ug/ml each, 1 mM PMSF) and phosphatase (1 mM NaVO4 ) inhibitors for 20 minutes on ice.

    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
    Article Snippet: .. After pretreatment with the ERK inhibitor U0126 (Calbiochem, Billerica, MA, USA) for 1 h and treatment with/without [6]-gingerol for 24 h, cells were harvested, and then protein was extracted with protein lysis buffer (25 mM Tris-Cl, pH 7.5, 250 mM NaCl, 5 mM ethylendiaminetetracetic acid, 1% Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails; Thermo Scientific, Waltham, MA, USA). .. Quantification of protein concentration was carried out using the Bradford method (Bio-Rad protein assay reagent), and total protein was resuspended in Laemmli sample buffer containing 5% β -mercaptoethanol and heated at 65°C for 10 min. Aliquots containing ~20–50 μ g of total cell proteins were resolved on 8–12% sodium dodecyl sulfate (SDS)-PAGE and then transferred onto nitrocellulose membranes (Amersham, Arlington Heights, IL, USA).

    FACS:

    Article Title: Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis
    Article Snippet: Naive T cells from spleen were purified by FACS of TCRβ+ /CD62Lhigh /CD44low cells. .. Naive T cells (5 × 105 ) were added to culture plates coated (or not) with various doses of anti-CD3 antibody (BD Biosciences) and 2 μg/mL anti-CD28 in the presence or absence of the ERK inhibitor U0126 (Sigma) and were cultured for 24 h. Supernatant was collected for assay of IL-2 using an ELISA kit (eBiosciences), and fresh medium was added.

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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: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of MEK and MAPK inhibitors on claudin-5 expression. Cells were treated with medium alone, 10 µM <t>U0126,</t> 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
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    Millipore mek erk inhibitor u0126
    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 <t>MEK/ERK</t> 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.
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    Millipore mapk extracellular signal regulated kinase kinase mek inhibitor u0126
    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), <t>MAPK/extracellular</t> signal‐regulated kinase kinase inhibitor <t>U0126</t> (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
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    Image Search Results


    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