p38 inhibitor sb202190  (Millipore)


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    Name:
    SB 202190
    Description:

    Catalog Number:
    s7067
    Price:
    None
    Applications:
    SB 202190 was used to inhibit p38 activation in MCF7 cells5, mouse macrophages6 and HepG2 cells.7
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    Structured Review

    Millipore p38 inhibitor sb202190
    SB 202190

    https://www.bioz.com/result/p38 inhibitor sb202190/product/Millipore
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    p38 inhibitor sb202190 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) 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

    2) Product Images from "Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model"

    Article Title: Inhibition of p38 MAPK attenuates renal atrophy and fibrosis in a murine renal artery stenosis model

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00706.2012

    Inhibition of p38 blocks TGF-β-induced upregulation of CCL2 and col4 expression in rat glomerular mesangial cells (MC). MC were pretreated with vehicle (0.1% DMSO) or p38 inhibitor SB202190 for 1 h, followed by 10 ng/ml TGF-β1 for 6 h. Total RNA was isolated and the copy number of each gene in 1 μg total RNA was measured by absolute quantitation real-time PCR and normalized by 18S rRNA. CCL2 ( A ); Col4a ( B ); CCR2 ( C ). Values are means ± SE, * P
    Figure Legend Snippet: Inhibition of p38 blocks TGF-β-induced upregulation of CCL2 and col4 expression in rat glomerular mesangial cells (MC). MC were pretreated with vehicle (0.1% DMSO) or p38 inhibitor SB202190 for 1 h, followed by 10 ng/ml TGF-β1 for 6 h. Total RNA was isolated and the copy number of each gene in 1 μg total RNA was measured by absolute quantitation real-time PCR and normalized by 18S rRNA. CCL2 ( A ); Col4a ( B ); CCR2 ( C ). Values are means ± SE, * P

    Techniques Used: Inhibition, Expressing, Isolation, Quantitation Assay, Real-time Polymerase Chain Reaction

    Inhibition of p38 reduces TNF-α-stimulated CCL2 expression in rat MC. MC were pretreated with vehicle or p38 inhibitor SB202190 for 1 h, followed by 10 ng/ml TNF-α for 6 h. The concentration of CCL2 secreted into the culture medium was quantified by using a rat-specific CCL2 ELISA kit. Values are means ± SE, * P
    Figure Legend Snippet: Inhibition of p38 reduces TNF-α-stimulated CCL2 expression in rat MC. MC were pretreated with vehicle or p38 inhibitor SB202190 for 1 h, followed by 10 ng/ml TNF-α for 6 h. The concentration of CCL2 secreted into the culture medium was quantified by using a rat-specific CCL2 ELISA kit. Values are means ± SE, * P

    Techniques Used: Inhibition, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Intestinal Epithelial Apoptosis initiates Gut Mucosal Injury during Extracorporeal Membrane Oxygenation in the Newborn Piglet"

    Article Title: Intestinal Epithelial Apoptosis initiates Gut Mucosal Injury during Extracorporeal Membrane Oxygenation in the Newborn Piglet

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2013.149

    Activation of p38 MAPK plays a key role in fas ligand production in mast cells in the ECMO intestine A. ECMO is associated with increased phospho-p38 expression in intestinal mast cells. Representative histograms obtained by FACS analysis show phospho-p38 expression in intestinal mast cells from sham and ECMO piglets, obtained after 2h of treatment. B. Fluorescent photomicrographs (630×) show that phospho-p38 (green) was immunolocalized in c-kit/CD117 + mast cells (red). Data represent n= 5 animals in sham and ECMO groups. C . Representative Western blots show that LPS-treatment of mast cells isolated from normal piglet intestine ex vivo promotes p38 phosphorylation in these cells. Bar diagram (means ± SE) shows summarized densitometric data. D . Bar diagram (means ± SE) shows fold change in fas ligand mRNA expression in intestinal mast cells (isolated from normal piglet intestine) in the native state, after treatment with LPS (1 μg/mL), and with SB202190 (1 μM) prior to LPS-treatment. Data represent 3 separate experiments. * indicates p
    Figure Legend Snippet: Activation of p38 MAPK plays a key role in fas ligand production in mast cells in the ECMO intestine A. ECMO is associated with increased phospho-p38 expression in intestinal mast cells. Representative histograms obtained by FACS analysis show phospho-p38 expression in intestinal mast cells from sham and ECMO piglets, obtained after 2h of treatment. B. Fluorescent photomicrographs (630×) show that phospho-p38 (green) was immunolocalized in c-kit/CD117 + mast cells (red). Data represent n= 5 animals in sham and ECMO groups. C . Representative Western blots show that LPS-treatment of mast cells isolated from normal piglet intestine ex vivo promotes p38 phosphorylation in these cells. Bar diagram (means ± SE) shows summarized densitometric data. D . Bar diagram (means ± SE) shows fold change in fas ligand mRNA expression in intestinal mast cells (isolated from normal piglet intestine) in the native state, after treatment with LPS (1 μg/mL), and with SB202190 (1 μM) prior to LPS-treatment. Data represent 3 separate experiments. * indicates p

    Techniques Used: Activation Assay, Expressing, FACS, Western Blot, Isolation, Ex Vivo

    4) Product Images from "Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation"

    Article Title: Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2014.132

    p38 and JNK1/2 pathways regulate resveratrol-induced H2AX phosphorylation. (A) K562 cells were treated with 60 μmol/L resveratrol, which was combined with the indicated concentration of the p38 inhibitor SB202190, for 24 h. The extracted whole
    Figure Legend Snippet: p38 and JNK1/2 pathways regulate resveratrol-induced H2AX phosphorylation. (A) K562 cells were treated with 60 μmol/L resveratrol, which was combined with the indicated concentration of the p38 inhibitor SB202190, for 24 h. The extracted whole

    Techniques Used: Concentration Assay

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

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.00001-17

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

    Techniques Used: Transfection, Western Blot, Plasmid Preparation

    6) Product Images from "The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU-Rich Element-Dependent Translation"

    Article Title: The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU-Rich Element-Dependent Translation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002977

    MK2-dependent translational control of TNF mRNA detected by ribosomal profiling after ER/cytosol cell fractionation of MK2-rescued and GFP-transduced macrophages and of wild type, MK2-deficient, and MK2/MK3 double-deficient primary BMDM. A) Polysome profile and fractions (1–12) within the profile. A schematic representation of the position of RNPs, ribosomal subunits, monosomes and polysomes is given above. Insert: TNF and ß-actin mRNA distibution (cytosol/ER ratio) after LPS-treatment of MK2-rescued (+MK2) or GFP-transduced (+GFP) macrophages. The difference in the TNF/18S is significant with p = 0.007. B) Distribution of TNF mRNA in the ER and cytosolic polysome profiles of MK2-rescued and GFP-transduced cells 1 h after LPS stimulation. An MK2-dependent redistribution of TNF mRNA to polysomal ER fractions (peaking in fraction 9) is observed. Rescue with the catalytic dead mutant MK2 K79R and block of MK2 activation by the p38 inhibitor SB202190 (MK2+SB202190) leads to a loss of the redistribution. C) MK2-independent distribution of ß-actin mRNA in the polysome profiles. D) MK2-independent distribution of the mRNA of the secreted cytokine KC/Cxcl1. E) Polysome profiles of total lysates of wild type, MK2-deficient and MK2/MK3 double-deficient primary BMDM. F,G) Distribution of TNF mRNA (F) and actin mRNA (G) in the polysome profile of WT, MK2-deficient and MK2/3-deficient primary macrophages.
    Figure Legend Snippet: MK2-dependent translational control of TNF mRNA detected by ribosomal profiling after ER/cytosol cell fractionation of MK2-rescued and GFP-transduced macrophages and of wild type, MK2-deficient, and MK2/MK3 double-deficient primary BMDM. A) Polysome profile and fractions (1–12) within the profile. A schematic representation of the position of RNPs, ribosomal subunits, monosomes and polysomes is given above. Insert: TNF and ß-actin mRNA distibution (cytosol/ER ratio) after LPS-treatment of MK2-rescued (+MK2) or GFP-transduced (+GFP) macrophages. The difference in the TNF/18S is significant with p = 0.007. B) Distribution of TNF mRNA in the ER and cytosolic polysome profiles of MK2-rescued and GFP-transduced cells 1 h after LPS stimulation. An MK2-dependent redistribution of TNF mRNA to polysomal ER fractions (peaking in fraction 9) is observed. Rescue with the catalytic dead mutant MK2 K79R and block of MK2 activation by the p38 inhibitor SB202190 (MK2+SB202190) leads to a loss of the redistribution. C) MK2-independent distribution of ß-actin mRNA in the polysome profiles. D) MK2-independent distribution of the mRNA of the secreted cytokine KC/Cxcl1. E) Polysome profiles of total lysates of wild type, MK2-deficient and MK2/MK3 double-deficient primary BMDM. F,G) Distribution of TNF mRNA (F) and actin mRNA (G) in the polysome profile of WT, MK2-deficient and MK2/3-deficient primary macrophages.

    Techniques Used: Cell Fractionation, Mutagenesis, Blocking Assay, Activation Assay

    7) Product Images from "Role of c-Jun N-Terminal Protein Kinase 1/2 (JNK1/2) in Macrophage-Mediated MMP-9 Production in Response to Moraxella catarrhalis Lipooligosaccharide (LOS)"

    Article Title: Role of c-Jun N-Terminal Protein Kinase 1/2 (JNK1/2) in Macrophage-Mediated MMP-9 Production in Response to Moraxella catarrhalis Lipooligosaccharide (LOS)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037912

    Inhibition of JNK1/2 by SP600125 increases LOS induced MMP-9 production in murine macrophages. A, RAW 264.7 cells were seeded in 96 well plate at 1×10 5 /100 µl/well. Cells were pretreated with AKT inhibitor LY 294002 (10 µM) and various MAPK inhibitors (ERK1/2 inhibitor U0126 (10 µM), p38 inhibitor SB202190 (10 µM) and JNK1/2 inhibitor SP600125 (10 µM) for 60 min and followed by LOS (100 ng/ml) treatment for 18 hrs. Supernatants were collected and activity of MMP-9 and MMP-2 were detected by zymogram (upper panel) and levels of MMP-9 were determined by ELISA (lower panel). B, RAW 264.7 cells were pretreated with different concentration of JNK1/2 inhibitor (0.1 µM, 1.0 µM, 5.0 µM and 10.0 µM) as indicated in figure followed by LOS treatment as described above. MMP-9 enzymatic activity and MMP-9 gene expression were determined by zymogram (upper panel) and real time-PCR, respectively (lower panel). C, Bone marrow derived macrophages were again pretreated with SP600125 (10 µM) for 1 hour followed by LOS treatment (100 ng/ml) for 48 hours. Cell culture supernatants were obtained and levels of MMP-9 were measured using ELISA kit. Typical results of three independent experiments are shown. ** ( p
    Figure Legend Snippet: Inhibition of JNK1/2 by SP600125 increases LOS induced MMP-9 production in murine macrophages. A, RAW 264.7 cells were seeded in 96 well plate at 1×10 5 /100 µl/well. Cells were pretreated with AKT inhibitor LY 294002 (10 µM) and various MAPK inhibitors (ERK1/2 inhibitor U0126 (10 µM), p38 inhibitor SB202190 (10 µM) and JNK1/2 inhibitor SP600125 (10 µM) for 60 min and followed by LOS (100 ng/ml) treatment for 18 hrs. Supernatants were collected and activity of MMP-9 and MMP-2 were detected by zymogram (upper panel) and levels of MMP-9 were determined by ELISA (lower panel). B, RAW 264.7 cells were pretreated with different concentration of JNK1/2 inhibitor (0.1 µM, 1.0 µM, 5.0 µM and 10.0 µM) as indicated in figure followed by LOS treatment as described above. MMP-9 enzymatic activity and MMP-9 gene expression were determined by zymogram (upper panel) and real time-PCR, respectively (lower panel). C, Bone marrow derived macrophages were again pretreated with SP600125 (10 µM) for 1 hour followed by LOS treatment (100 ng/ml) for 48 hours. Cell culture supernatants were obtained and levels of MMP-9 were measured using ELISA kit. Typical results of three independent experiments are shown. ** ( p

    Techniques Used: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Cell Culture

    8) Product Images from "Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation"

    Article Title: Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2014.132

    p38 and JNK1/2 pathways regulate resveratrol-induced H2AX phosphorylation. (A) K562 cells were treated with 60 μmol/L resveratrol, which was combined with the indicated concentration of the p38 inhibitor SB202190, for 24 h. The extracted whole cellular protein samples were subjected to Western blot analysis with antibodies against p-p38 and total p38, and the histone samples were used for Western blot analysis with antibodies against γH2AX and total H2AX. (B) K562 cells were treated with 60 μmol/L resveratrol, which was combined with the indicated concentration of the JNK inhibitor SP600125, for 24 h. The whole cellular protein samples were evaluated for pJNK1/2 and total JNK1/2, and the histone protein samples were evaluated for γH2AX and total H2AX. β-Actin was used to confirm equal whole cellular protein loading. (C) After being transfected with p38-specific siRNA (100 nmol/L), JNK-specific siRNA (100 nmol/L) or control siRNA (100 nmol/L), the K562 cells were treated with 60 μmol/L resveratrol for 24 h. The histone samples were extracted to detect γH2AX and total H2AX, and the extracted whole cellular protein samples were used to detect the effects of p38 and JNK knockdown. β-Actin was used to confirm equal whole cellular protein loading.
    Figure Legend Snippet: p38 and JNK1/2 pathways regulate resveratrol-induced H2AX phosphorylation. (A) K562 cells were treated with 60 μmol/L resveratrol, which was combined with the indicated concentration of the p38 inhibitor SB202190, for 24 h. The extracted whole cellular protein samples were subjected to Western blot analysis with antibodies against p-p38 and total p38, and the histone samples were used for Western blot analysis with antibodies against γH2AX and total H2AX. (B) K562 cells were treated with 60 μmol/L resveratrol, which was combined with the indicated concentration of the JNK inhibitor SP600125, for 24 h. The whole cellular protein samples were evaluated for pJNK1/2 and total JNK1/2, and the histone protein samples were evaluated for γH2AX and total H2AX. β-Actin was used to confirm equal whole cellular protein loading. (C) After being transfected with p38-specific siRNA (100 nmol/L), JNK-specific siRNA (100 nmol/L) or control siRNA (100 nmol/L), the K562 cells were treated with 60 μmol/L resveratrol for 24 h. The histone samples were extracted to detect γH2AX and total H2AX, and the extracted whole cellular protein samples were used to detect the effects of p38 and JNK knockdown. β-Actin was used to confirm equal whole cellular protein loading.

    Techniques Used: Concentration Assay, Western Blot, Transfection

    9) Product Images from "Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation"

    Article Title: Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004393

    Effect of MAP kinase and MSK1 inhibitors on IL-1β-induced p65, MSK1 and CBP binding to the κB site of the SCF intronic enhancer. Human lung fibroblasts in culture were pre-incubated for 1 h with a combination of the p38 inhibitor SB202190 (SB; 3.5 µM) and the MEK inhibitor PD98059 (PD; 20 µM) or with the MSK1-PKA inhibitor H89 (10 µM) and treated with IL-1β (20 U/ml) for 30 min. The ChIP experiment was performed with anti-p65, MSK1, CBP, phospho-Ser10 histone H3 and control Ig antibodies. Co-immunoprecipitated genomic DNA fragments were amplified by PCR with SCF intronic enhancer-specific primers. Input reflects the relative amounts of sonicated DNA fragments before immunoprecipitation. Results are representative of 3 independent experiments performed in fibroblasts from 3 different donors.
    Figure Legend Snippet: Effect of MAP kinase and MSK1 inhibitors on IL-1β-induced p65, MSK1 and CBP binding to the κB site of the SCF intronic enhancer. Human lung fibroblasts in culture were pre-incubated for 1 h with a combination of the p38 inhibitor SB202190 (SB; 3.5 µM) and the MEK inhibitor PD98059 (PD; 20 µM) or with the MSK1-PKA inhibitor H89 (10 µM) and treated with IL-1β (20 U/ml) for 30 min. The ChIP experiment was performed with anti-p65, MSK1, CBP, phospho-Ser10 histone H3 and control Ig antibodies. Co-immunoprecipitated genomic DNA fragments were amplified by PCR with SCF intronic enhancer-specific primers. Input reflects the relative amounts of sonicated DNA fragments before immunoprecipitation. Results are representative of 3 independent experiments performed in fibroblasts from 3 different donors.

    Techniques Used: Binding Assay, Incubation, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Sonication

    10) Product Images from "Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling"

    Article Title: Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00781-17

    CD14-dependent activation of p38 is required for IL-10 production. (A) Equal proteins from lysates of CD14 +/+ and CD14 −/− B6 BMDMs incubated with B. burgdorferi for 30 min were separated by 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed with phospho-p38 and β-actin antibodies. CD14 +/+ BMDMs (B) or PMA-treated THP-1 cells (C) were treated with DMSO or increasing concentrations of SB202190 for 30 min prior to incubation with B. burgdorferi . Cell culture supernatants were collected 24 h postinfection, and TNF and IL-10 levels were measured by ELISA. (D) CD14 +/+ BMDMs were pretreated with combinations of DMSO, 5 μM SB202190, and 10, 50, or 100 ng/ml of recombinant IL-10 and then coincubated with B. burgdorferi ( B.b .) at an MOI of 10. TNF was measured in the culture supernatants collected after 24 h. (E) B. burgdorferi organisms were coincubated with PBMCs from 10 patients with Lyme arthritis, 5 with the TLR1-1805GG SNP and 5 with the TLR1-1805TG/TT SNP, for 30 min. Cell lysates were analyzed for phospho-p38 by the bead-based Luminex assay (Millipore). For panel A, data are representative of results from two independent experiments. For panels B and C, data are representative of results from five independent experiments. For panels D and E, results represent means ± SEMs from two independent experiments. ** , P
    Figure Legend Snippet: CD14-dependent activation of p38 is required for IL-10 production. (A) Equal proteins from lysates of CD14 +/+ and CD14 −/− B6 BMDMs incubated with B. burgdorferi for 30 min were separated by 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed with phospho-p38 and β-actin antibodies. CD14 +/+ BMDMs (B) or PMA-treated THP-1 cells (C) were treated with DMSO or increasing concentrations of SB202190 for 30 min prior to incubation with B. burgdorferi . Cell culture supernatants were collected 24 h postinfection, and TNF and IL-10 levels were measured by ELISA. (D) CD14 +/+ BMDMs were pretreated with combinations of DMSO, 5 μM SB202190, and 10, 50, or 100 ng/ml of recombinant IL-10 and then coincubated with B. burgdorferi ( B.b .) at an MOI of 10. TNF was measured in the culture supernatants collected after 24 h. (E) B. burgdorferi organisms were coincubated with PBMCs from 10 patients with Lyme arthritis, 5 with the TLR1-1805GG SNP and 5 with the TLR1-1805TG/TT SNP, for 30 min. Cell lysates were analyzed for phospho-p38 by the bead-based Luminex assay (Millipore). For panel A, data are representative of results from two independent experiments. For panels B and C, data are representative of results from five independent experiments. For panels D and E, results represent means ± SEMs from two independent experiments. ** , P

    Techniques Used: Activation Assay, Incubation, SDS Page, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Luminex

    p38 regulates binding of transcription factors STAT-3 and SP-1 to the il-10 promoter in B6 BMDMs. BMDMs were coincubated with B. burgdorferi at an MOI of 10 for 30 min in the presence or absence of 5 μM SB202190. Subsequently, a ChIP assay was performed to evaluate the binding of STAT3 to the il10 promoter at the −700 site (A) or −100 site (B). The data were normalized to the input DNA and are shown as fold enrichment over uninfected controls. (C) A similar strategy was used to evaluate the binding of SP1 to the i10 promoter. Results represent means ± SEMs from two independent experiments. The dotted line represents a 1.5-fold change, considered a significant change from the control. ** , P
    Figure Legend Snippet: p38 regulates binding of transcription factors STAT-3 and SP-1 to the il-10 promoter in B6 BMDMs. BMDMs were coincubated with B. burgdorferi at an MOI of 10 for 30 min in the presence or absence of 5 μM SB202190. Subsequently, a ChIP assay was performed to evaluate the binding of STAT3 to the il10 promoter at the −700 site (A) or −100 site (B). The data were normalized to the input DNA and are shown as fold enrichment over uninfected controls. (C) A similar strategy was used to evaluate the binding of SP1 to the i10 promoter. Results represent means ± SEMs from two independent experiments. The dotted line represents a 1.5-fold change, considered a significant change from the control. ** , P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    11) Product Images from "Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells"

    Article Title: Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells

    Journal: FEMS immunology and medical microbiology

    doi: 10.1111/j.1574-695X.2008.00439.x

    Effects of different MAPK inhibitors on AP-1 DNA binding activity in AGS cells. AGS cells (5×10 5 ) were pre-incubated with MEK1/2 inhibitor PD 98059 (PD), p38 inhibitor SB202190 (SB), and JNK inhibitor SP600125 (SP) at 10 μM dose for 30 minutes and then treated in the presence or absence of wild-type H. pylori 26695 (HP) at an MOI of 150:1 in antibiotic-free Ham’s F-12 medium without FBS for 6 hours. EMSA assay (A), or supershift assay (B) were performed as described in Materials and Methods. Photos are representative of two to three separate experiments with similar results, data are mean values from densitometry scans, and expressed as fold changes over the appropriate control. “SS”, super shift assay; “ ← ” define AP-1
    Figure Legend Snippet: Effects of different MAPK inhibitors on AP-1 DNA binding activity in AGS cells. AGS cells (5×10 5 ) were pre-incubated with MEK1/2 inhibitor PD 98059 (PD), p38 inhibitor SB202190 (SB), and JNK inhibitor SP600125 (SP) at 10 μM dose for 30 minutes and then treated in the presence or absence of wild-type H. pylori 26695 (HP) at an MOI of 150:1 in antibiotic-free Ham’s F-12 medium without FBS for 6 hours. EMSA assay (A), or supershift assay (B) were performed as described in Materials and Methods. Photos are representative of two to three separate experiments with similar results, data are mean values from densitometry scans, and expressed as fold changes over the appropriate control. “SS”, super shift assay; “ ← ” define AP-1

    Techniques Used: Binding Assay, Activity Assay, Incubation, Super-Shift Assay

    Effects of H. pylori and MAPK inhibition on AP-1 proteins expression in AGS cells. AGS cell (5×10 5 ) were treated in the presence or absence of wild-type H. pylori 26695 at an MOI of 150:1, and with different doses of MAPK inhibitors (PD98059 (PD) for ERK, SB202190 (SB) for p38, and SP600190 (SP) for JNK) in antibiotic-free Ham’s F-12 medium without FBS for 6 hours. Control cells were treated with medium alone. The cells were and resolved on a 12% SDS-PAGE, and incubated with polyclonal antibodies for human c-Jun, JunB, c-Fos, Fra-1 proteins on nitrocellulose membrane. The original membrane was stripped and re-probed with β-actin antibody to monitor protein loading. Blots are representative of two to three separate experiments with similar results. Data are mean values from densitometry scans, adjusted with β-actin, and expressed as fold changes over the appropriate control.
    Figure Legend Snippet: Effects of H. pylori and MAPK inhibition on AP-1 proteins expression in AGS cells. AGS cell (5×10 5 ) were treated in the presence or absence of wild-type H. pylori 26695 at an MOI of 150:1, and with different doses of MAPK inhibitors (PD98059 (PD) for ERK, SB202190 (SB) for p38, and SP600190 (SP) for JNK) in antibiotic-free Ham’s F-12 medium without FBS for 6 hours. Control cells were treated with medium alone. The cells were and resolved on a 12% SDS-PAGE, and incubated with polyclonal antibodies for human c-Jun, JunB, c-Fos, Fra-1 proteins on nitrocellulose membrane. The original membrane was stripped and re-probed with β-actin antibody to monitor protein loading. Blots are representative of two to three separate experiments with similar results. Data are mean values from densitometry scans, adjusted with β-actin, and expressed as fold changes over the appropriate control.

    Techniques Used: Inhibition, Expressing, SDS Page, Incubation

    12) Product Images from "Shiga Toxin 1 Triggers a Ribotoxic Stress Response Leading to p38 and JNK Activation and Induction of Apoptosis in Intestinal Epithelial Cells "

    Article Title: Shiga Toxin 1 Triggers a Ribotoxic Stress Response Leading to p38 and JNK Activation and Induction of Apoptosis in Intestinal Epithelial Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.71.3.1497-1504.2003

    Effect of SB202190 on Stx1-induced cell death and Stx1-induced p38 and JNK activation. (A) After treatment of HCT-8 cells for 72 h with various concentrations of Stx1 or Stx1E167D, cell viability was measured by MTT assay expressed as percentage of control cell viability. (B) Effects of various doses of SB202190 on Stx1-induced p38 and JNK activation in HCT-8 cells. Briefly, cells were preincubated with doses of SB202190 ranging from 3 to 48 μM, or equal amounts of the SB202190 diluent DMSO, and then exposed to Stx1 at 1 μg/ml. Cell extracts were obtained and assessments of p38 and JNK activation were performed as outlined in Materials and Methods. (C) After treatment of HCT-8 cells for 48 h with various concentrations of Stx1 with and without the p38 inhibitor SB202190, cell viability was measured by MTT assay and expressed as percentage of control cell viability. In panels A and C, optical density measurements for control cells were averaged, and then the optical density (at 540 nm) reading for each well in the microtiter plate was expressed as a percentage of the control average, including the individual control measurements. The percentages were then averaged for each group, and data are expressed as a mean ± standard deviation. For each condition, n = 3 wells. In panels A and C, * denotes a P value of
    Figure Legend Snippet: Effect of SB202190 on Stx1-induced cell death and Stx1-induced p38 and JNK activation. (A) After treatment of HCT-8 cells for 72 h with various concentrations of Stx1 or Stx1E167D, cell viability was measured by MTT assay expressed as percentage of control cell viability. (B) Effects of various doses of SB202190 on Stx1-induced p38 and JNK activation in HCT-8 cells. Briefly, cells were preincubated with doses of SB202190 ranging from 3 to 48 μM, or equal amounts of the SB202190 diluent DMSO, and then exposed to Stx1 at 1 μg/ml. Cell extracts were obtained and assessments of p38 and JNK activation were performed as outlined in Materials and Methods. (C) After treatment of HCT-8 cells for 48 h with various concentrations of Stx1 with and without the p38 inhibitor SB202190, cell viability was measured by MTT assay and expressed as percentage of control cell viability. In panels A and C, optical density measurements for control cells were averaged, and then the optical density (at 540 nm) reading for each well in the microtiter plate was expressed as a percentage of the control average, including the individual control measurements. The percentages were then averaged for each group, and data are expressed as a mean ± standard deviation. For each condition, n = 3 wells. In panels A and C, * denotes a P value of

    Techniques Used: Activation Assay, MTT Assay, Standard Deviation

    13) Product Images from "Tumor necrosis factor α modulates sodium-activated potassium channel SLICK in rat dorsal horn neurons via p38 MAPK activation pathway"

    Article Title: Tumor necrosis factor α modulates sodium-activated potassium channel SLICK in rat dorsal horn neurons via p38 MAPK activation pathway

    Journal: Journal of Pain Research

    doi: 10.2147/JPR.S132185

    The K Na current is significantly decreased in DH neurons treated with TNF-α via p38 MAPK. Notes: ( A, B ) Left, Representative traces of I K recorded before and after application of 10 ng/mL TNF-α in 140 and 0 mM Na + external solution; Right, Current–voltage relationships before and after TNF-α treatment. ( C, D ) Left, Representative traces of I K recorded before and after application of 10 μM of p38 inhibitor SB202190 for 30 min and then 10 min TNF-α treatment in 140 mM and 0 mM Na + external solution; Right, Current–voltage relationships before and after SB202190 and then TNF-α treatment. ( E, F ) Peak current after 10 ng/mL TNF-α and TNF-α combined SB202190 addition at 50 mV normalized to control for both 140 and 0 mM Na-containing extracellular solution. * P
    Figure Legend Snippet: The K Na current is significantly decreased in DH neurons treated with TNF-α via p38 MAPK. Notes: ( A, B ) Left, Representative traces of I K recorded before and after application of 10 ng/mL TNF-α in 140 and 0 mM Na + external solution; Right, Current–voltage relationships before and after TNF-α treatment. ( C, D ) Left, Representative traces of I K recorded before and after application of 10 μM of p38 inhibitor SB202190 for 30 min and then 10 min TNF-α treatment in 140 mM and 0 mM Na + external solution; Right, Current–voltage relationships before and after SB202190 and then TNF-α treatment. ( E, F ) Peak current after 10 ng/mL TNF-α and TNF-α combined SB202190 addition at 50 mV normalized to control for both 140 and 0 mM Na-containing extracellular solution. * P

    Techniques Used:

    TNF-α decreases membrane expression of SLICK channels via p38 MAPK through possible posttranslational modification in DH neurons. Notes: ( A ) Representative blot of membrane SLICK biotinylation assay. Membrane biotinylation and precipitation by streptavidin followed by Western analysis using a SLICK-specific antibody were performed on untreated neurons, neurons treated with 10 ng/mL TNF-α for 10 min, or neurons pretreated with 10 μM of p38 inhibitor SB202190 for 30 min followed by 10 ng/mL TNF-α for 10 min. ( B ) Densitometric analysis of SLICK membrane expression when normalized to β-actin. ( C ) Normalized SLICK gene expression levels. * P
    Figure Legend Snippet: TNF-α decreases membrane expression of SLICK channels via p38 MAPK through possible posttranslational modification in DH neurons. Notes: ( A ) Representative blot of membrane SLICK biotinylation assay. Membrane biotinylation and precipitation by streptavidin followed by Western analysis using a SLICK-specific antibody were performed on untreated neurons, neurons treated with 10 ng/mL TNF-α for 10 min, or neurons pretreated with 10 μM of p38 inhibitor SB202190 for 30 min followed by 10 ng/mL TNF-α for 10 min. ( B ) Densitometric analysis of SLICK membrane expression when normalized to β-actin. ( C ) Normalized SLICK gene expression levels. * P

    Techniques Used: Expressing, Modification, Cell Surface Biotinylation Assay, Western Blot

    The cell cytotoxicity of TNF-α and the effect of p38 inhibitor SB202190 on the viability of DH neuron were evaluated using MTT assay. Notes: MTT assay that SB202190 in combination with TNF-α exerted no effect on the viability of DH neurons. Abbreviations: DH, dorsal horn; TNF-α, tumor necrosis factor-alpha.
    Figure Legend Snippet: The cell cytotoxicity of TNF-α and the effect of p38 inhibitor SB202190 on the viability of DH neuron were evaluated using MTT assay. Notes: MTT assay that SB202190 in combination with TNF-α exerted no effect on the viability of DH neurons. Abbreviations: DH, dorsal horn; TNF-α, tumor necrosis factor-alpha.

    Techniques Used: MTT Assay

    SB202190 restores the firing accommodation of DH neurons. Notes: ( A ) Representative action potential recorded in untreated neurons. ( B ) 5 min application of 10 ng/mL TNF-α, neurons exhibited a progressive loss of firing accommodation. ( C ) 10 min application of 10 ng/mL TNF-α, neurons showed completed loss of firing accommodation. ( D ) DH neurons were incubated with 10 μM of p38 inhibitor SB202190 for 30 min before TNF-α treatment; no loss of firing accommodation was detected. Using whole-cell current-clamp on cultured DH neurons, stimulation was 2.5× threshold for 1000 ms. Abbreviation: DH, dorsal horn.
    Figure Legend Snippet: SB202190 restores the firing accommodation of DH neurons. Notes: ( A ) Representative action potential recorded in untreated neurons. ( B ) 5 min application of 10 ng/mL TNF-α, neurons exhibited a progressive loss of firing accommodation. ( C ) 10 min application of 10 ng/mL TNF-α, neurons showed completed loss of firing accommodation. ( D ) DH neurons were incubated with 10 μM of p38 inhibitor SB202190 for 30 min before TNF-α treatment; no loss of firing accommodation was detected. Using whole-cell current-clamp on cultured DH neurons, stimulation was 2.5× threshold for 1000 ms. Abbreviation: DH, dorsal horn.

    Techniques Used: Incubation, Cell Culture, Mass Spectrometry

    14) Product Images from "A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro"

    Article Title: A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2016.2300

    Dovitinib activates MAPK signaling pathways in BMP-2-induced osteoblast differentiation. The C2C12 cells were treated with BMP-2 (50 ng/ml) ± dovitinib for 10 minutes. (A) The phosphorylation of ERK1/2 and p38 was measured by Western blot analysis. Actin was used as a loading control. (B, C) The C2C12 cells were treated with PD98059 or SB202190 in the presence of BMP-2 (50 ng/ml) and dovitinib (100 nM) for 3 days. The effect of inhibitors on the BMP-2-induced osteoblast differentiation was detected by the induction of ALP activity in C2C12 cells. Experiments were performed in triplicate. Scale bars represent 100 μm.
    Figure Legend Snippet: Dovitinib activates MAPK signaling pathways in BMP-2-induced osteoblast differentiation. The C2C12 cells were treated with BMP-2 (50 ng/ml) ± dovitinib for 10 minutes. (A) The phosphorylation of ERK1/2 and p38 was measured by Western blot analysis. Actin was used as a loading control. (B, C) The C2C12 cells were treated with PD98059 or SB202190 in the presence of BMP-2 (50 ng/ml) and dovitinib (100 nM) for 3 days. The effect of inhibitors on the BMP-2-induced osteoblast differentiation was detected by the induction of ALP activity in C2C12 cells. Experiments were performed in triplicate. Scale bars represent 100 μm.

    Techniques Used: Western Blot, ALP Assay, Activity Assay

    15) Product Images from "A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis *"

    Article Title: A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.438010

    The EGFR inhibitor blocks PV IgG-induced keratin intermediate filament retraction. Confocal immunofluorescent images of keratinocytes treated with NH or PV IgG ± the p38 inhibitor SB202190 or the EGFR inhibitor AG1478. Primary human keratinocytes
    Figure Legend Snippet: The EGFR inhibitor blocks PV IgG-induced keratin intermediate filament retraction. Confocal immunofluorescent images of keratinocytes treated with NH or PV IgG ± the p38 inhibitor SB202190 or the EGFR inhibitor AG1478. Primary human keratinocytes

    Techniques Used:

    Related Articles

    Incubation:

    Article Title: Brx Mediates the Response of Lymphocytes to Osmotic Stress Through the Activation of NFAT5
    Article Snippet: .. In some experiments, Jurkat cells were incubated with the p38 MAPK inhibitor SB202190 (2 μM) or the MEK1 inhibitor PD98059 (10 μM) (Sigma-Aldrich) in the absence or presence of 100 mM NaCl. .. After 8 hours of incubation, total RNA was harvested for real-time RT-PCR analysis.

    Recombinant:

    Article Title: Expression of leukemia inhibitory factor in Müller glia cells is regulated by a redox-dependent mRNA stability mechanism
    Article Snippet: .. Rat recombinant TNF (R & D systems, Minneapolis, MN, USA) and the p38 MAPK inhibitor SB202190 (Sigma Aldrich) [ ] were dissolved in 0.1% bovine serum albumin or in dimethyl sulfoxide (DMSO), respectively. ..

    Inhibition:

    Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis
    Article Snippet: .. ERK and p38 pharmacological inhibition 6 × 105 cells were plated in six well plates and pretreated with the ERK inhibitor SHC772984 (Cayman Chemical, Ann Arbor, MI, USA) or the p38 inhibitor SB 202190 (Sigma-Aldrich) for 18 hr, using the following concentrations: 30, 100 or 300 nM for ERK inhibitor, and 10, 100 or 300 μM for p38 inhibitor. ..

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    Millipore sb202190
    Fenretinide upregulation of DR is dependent on phosphorylation of ASK1, and p38 α . Ewing's sarcoma family of tumour cells were pretreated with ( A ) <t>SB202190</t> (20 μ , 1 h) or ( B ) BIRB0796 (0.1 or 1 μ , 24 h) before fenretinide treatment (3 μ , 24 h) and DR expression was determined by antibody labelling and flow cytometry. Results are presented as the fold increase of the mean of the receptor median fluorescence intensity relative to untreated control samples±s.e.m. ( n =6). ( Ci ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n), and ASK1 kinase activity was assessed 48 h later by immunecomplex kinase assays using MBP as the substrate. Samples were subsequently resolved by SDS–PAGE and gels were analysed by quantitative autoradiography using a PhosphorImager system. Untreated and 6-OHDA (100 μ , 1 h)-treated SH-SY5Y cells served as negative and positive controls, respectively, for ASK1 kinase activity. Graph shows ASK1 activity calculated as fold increase over untreated scrambled siRNA cells. ( Cii ) TC-32 cells were pretreated with vitamin C (100 μ , 1 h) before fenretinide treatment (3 μ , 10 min) and ASK1 immunecomplex kinase assays were performed. ( Ciii ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n). Protein expression was determined 48 h later by immunoblot analysis for total p38 MAPK and phospho-p38 MAPK and total MKK3 and phospho-MKK3/6. Anisomycin (25 μ g/ml, 30 min)-treated TC-32 cells served as a positive control for MKK3/6 phosphorylation. Equal protein loading was confirmed by hybridisation to tubulin. M=molecular weight markers. Representative images from three independent experiments are shown. ( D ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n) and were treated with fenretinide (3 μ , 24 h) 24 h after electroporation. DR expression was determined by antibody labelling and flow cytometry, and results presented as the fold increase of the mean of the receptor median fluorescence intensity relative to untreated control samples±s.e.m. ( n =6). * P ⩽0.001, ** P ⩽0.01, *** P ⩽0.05.
    Sb202190, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fenretinide upregulation of DR is dependent on phosphorylation of ASK1, and p38 α . Ewing's sarcoma family of tumour cells were pretreated with ( A ) SB202190 (20 μ , 1 h) or ( B ) BIRB0796 (0.1 or 1 μ , 24 h) before fenretinide treatment (3 μ , 24 h) and DR expression was determined by antibody labelling and flow cytometry. Results are presented as the fold increase of the mean of the receptor median fluorescence intensity relative to untreated control samples±s.e.m. ( n =6). ( Ci ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n), and ASK1 kinase activity was assessed 48 h later by immunecomplex kinase assays using MBP as the substrate. Samples were subsequently resolved by SDS–PAGE and gels were analysed by quantitative autoradiography using a PhosphorImager system. Untreated and 6-OHDA (100 μ , 1 h)-treated SH-SY5Y cells served as negative and positive controls, respectively, for ASK1 kinase activity. Graph shows ASK1 activity calculated as fold increase over untreated scrambled siRNA cells. ( Cii ) TC-32 cells were pretreated with vitamin C (100 μ , 1 h) before fenretinide treatment (3 μ , 10 min) and ASK1 immunecomplex kinase assays were performed. ( Ciii ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n). Protein expression was determined 48 h later by immunoblot analysis for total p38 MAPK and phospho-p38 MAPK and total MKK3 and phospho-MKK3/6. Anisomycin (25 μ g/ml, 30 min)-treated TC-32 cells served as a positive control for MKK3/6 phosphorylation. Equal protein loading was confirmed by hybridisation to tubulin. M=molecular weight markers. Representative images from three independent experiments are shown. ( D ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n) and were treated with fenretinide (3 μ , 24 h) 24 h after electroporation. DR expression was determined by antibody labelling and flow cytometry, and results presented as the fold increase of the mean of the receptor median fluorescence intensity relative to untreated control samples±s.e.m. ( n =6). * P ⩽0.001, ** P ⩽0.01, *** P ⩽0.05.

    Journal: British Journal of Cancer

    Article Title: Fenretinide-dependent upregulation of death receptors through ASK1 and p38α enhances death receptor ligand-induced cell death in Ewing's sarcoma family of tumours

    doi: 10.1038/sj.bjc.6605896

    Figure Lengend Snippet: Fenretinide upregulation of DR is dependent on phosphorylation of ASK1, and p38 α . Ewing's sarcoma family of tumour cells were pretreated with ( A ) SB202190 (20 μ , 1 h) or ( B ) BIRB0796 (0.1 or 1 μ , 24 h) before fenretinide treatment (3 μ , 24 h) and DR expression was determined by antibody labelling and flow cytometry. Results are presented as the fold increase of the mean of the receptor median fluorescence intensity relative to untreated control samples±s.e.m. ( n =6). ( Ci ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n), and ASK1 kinase activity was assessed 48 h later by immunecomplex kinase assays using MBP as the substrate. Samples were subsequently resolved by SDS–PAGE and gels were analysed by quantitative autoradiography using a PhosphorImager system. Untreated and 6-OHDA (100 μ , 1 h)-treated SH-SY5Y cells served as negative and positive controls, respectively, for ASK1 kinase activity. Graph shows ASK1 activity calculated as fold increase over untreated scrambled siRNA cells. ( Cii ) TC-32 cells were pretreated with vitamin C (100 μ , 1 h) before fenretinide treatment (3 μ , 10 min) and ASK1 immunecomplex kinase assays were performed. ( Ciii ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n). Protein expression was determined 48 h later by immunoblot analysis for total p38 MAPK and phospho-p38 MAPK and total MKK3 and phospho-MKK3/6. Anisomycin (25 μ g/ml, 30 min)-treated TC-32 cells served as a positive control for MKK3/6 phosphorylation. Equal protein loading was confirmed by hybridisation to tubulin. M=molecular weight markers. Representative images from three independent experiments are shown. ( D ) TC-32 cells were electroporated with scrambled or ASK1 siRNA (500 n) and were treated with fenretinide (3 μ , 24 h) 24 h after electroporation. DR expression was determined by antibody labelling and flow cytometry, and results presented as the fold increase of the mean of the receptor median fluorescence intensity relative to untreated control samples±s.e.m. ( n =6). * P ⩽0.001, ** P ⩽0.01, *** P ⩽0.05.

    Article Snippet: Fenretinide (gift from the National Cancer Institute), BAY 11-70892 (Calbiochem, Nottingham, UK) and SB202190 (Calbiochem) were prepared as previously described ( ; ).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence, Activity Assay, SDS Page, Autoradiography, Positive Control, Hybridization, Molecular Weight, Electroporation

    Inhibition of LH-induced connexin-43 phosphorylation by the EGFR inhibitor AG1478 and the p38MAPK inhibitor SB202190. A and B, LH-induced Cx43 phosphorylation on S262 and S255 was examined by Western blot analyses using protein extracts from preovulatory follicles that were pre-incubated for 30 min with or without the EGFR tyrosine kinase inhibitor AG1478 (0.5 µM), and then stimulated for 2 h with rLH (5 IU). The ratio of phosphorylated S262 or S255 to total Cx43 was normalized to the control and expressed as fold induction. Data are the mean ± range of two separate experiments, and are shown in the immunoblots. C and D, Western blot analyses to detect LH-induced phosphorylation of Cx43 on S262 and S255 was performed using protein extracts from preovulatory follicles pre-incubated for 30 min with or without SB202190 (10 µM) and stimulated for 2 h with rLH (5 IU). The ratio of phosphorylated S262 or S255 to total Cx43 was normalized to the control and expressed as fold induction. Data are the mean ± SEM of three separate experiments. Representative blots are shown.

    Journal: PLoS ONE

    Article Title: Genetic Dissection of Epidermal Growth Factor Receptor Signaling during Luteinizing Hormone-Induced Oocyte Maturation

    doi: 10.1371/journal.pone.0021574

    Figure Lengend Snippet: Inhibition of LH-induced connexin-43 phosphorylation by the EGFR inhibitor AG1478 and the p38MAPK inhibitor SB202190. A and B, LH-induced Cx43 phosphorylation on S262 and S255 was examined by Western blot analyses using protein extracts from preovulatory follicles that were pre-incubated for 30 min with or without the EGFR tyrosine kinase inhibitor AG1478 (0.5 µM), and then stimulated for 2 h with rLH (5 IU). The ratio of phosphorylated S262 or S255 to total Cx43 was normalized to the control and expressed as fold induction. Data are the mean ± range of two separate experiments, and are shown in the immunoblots. C and D, Western blot analyses to detect LH-induced phosphorylation of Cx43 on S262 and S255 was performed using protein extracts from preovulatory follicles pre-incubated for 30 min with or without SB202190 (10 µM) and stimulated for 2 h with rLH (5 IU). The ratio of phosphorylated S262 or S255 to total Cx43 was normalized to the control and expressed as fold induction. Data are the mean ± SEM of three separate experiments. Representative blots are shown.

    Article Snippet: Pregnant Mare Serum Gonadotropin (PMSG), AG1478 and SB202190 were purchased from Calbiochem (San Diego, CA).

    Techniques: Inhibition, Western Blot, Incubation

    Reversal of resistance of 40AF to 1,25D-induced differentiation is associated with increased expression of CYP24. HL60-G and 40AF cells were treated for 96 h with 10 nM 1,25D (D3) in combination with potentiators of 1,25D-induced differentiation, SB202190, and one of three plant-derived antioxidants, carnosic acid (DCS), silibinin (DSS), or curcumin (DCuS). A: FACS analysis of monocytic surface markers indicates a significant increase in CD11b and CD14 expression on HL60-G and 40AF cells treated with these triple combinations. Bars represent mean values + SEM (n = 5). B: The levels of VDR-induced transcription were determined by qRT-PCR of CYP24 mRNA levels and relative fold values were calculated as described in the Materials and Methods section. Treatment with triple combinations significantly enhanced the expression of VDR-target gene, CYP24 in 40AF cells when compared to vehicle-treated samples. Levels of CYP24 mRNA induced by DCS-treatment of HL60-G cells are shown as a positive control. ** signifies P

    Journal: Journal of cellular physiology

    Article Title: Resistance to 1,25D-Induced Differentiation in Human Acute Myeloid Leukemia HL60-40AF Cells Is Associated With Reduced Transcriptional Activity and Nuclear Localization of the Vitamin D Receptor

    doi: 10.1002/jcp.21150

    Figure Lengend Snippet: Reversal of resistance of 40AF to 1,25D-induced differentiation is associated with increased expression of CYP24. HL60-G and 40AF cells were treated for 96 h with 10 nM 1,25D (D3) in combination with potentiators of 1,25D-induced differentiation, SB202190, and one of three plant-derived antioxidants, carnosic acid (DCS), silibinin (DSS), or curcumin (DCuS). A: FACS analysis of monocytic surface markers indicates a significant increase in CD11b and CD14 expression on HL60-G and 40AF cells treated with these triple combinations. Bars represent mean values + SEM (n = 5). B: The levels of VDR-induced transcription were determined by qRT-PCR of CYP24 mRNA levels and relative fold values were calculated as described in the Materials and Methods section. Treatment with triple combinations significantly enhanced the expression of VDR-target gene, CYP24 in 40AF cells when compared to vehicle-treated samples. Levels of CYP24 mRNA induced by DCS-treatment of HL60-G cells are shown as a positive control. ** signifies P

    Article Snippet: Inhibitor of p38 MAPK, SB202190, was obtained from Calbiochem (La Jolla, CA), carnosic acid from Alexis Pharmaceuticals (Lausen, Switzerland), silibinin and curcumin from Sigma–Aldrich (St. Louis, MO).

    Techniques: Expressing, Derivative Assay, FACS, Quantitative RT-PCR, Positive Control

    Effect of MAPK inhibitors on ICAM-1 and VCAM-1 expressions. ( a ) HT29 cells were pretreated with SB202190 or SP600125 (10 μ M ) for 30 min and then incubated from time 0 to 8 h. At the time indicated, cells were processed

    Journal:

    Article Title: Celecoxib decreases expression of the adhesion molecules ICAM-1 and VCAM-1 in a colon cancer cell line (HT29)

    doi: 10.1038/sj.bjp.0707634

    Figure Lengend Snippet: Effect of MAPK inhibitors on ICAM-1 and VCAM-1 expressions. ( a ) HT29 cells were pretreated with SB202190 or SP600125 (10 μ M ) for 30 min and then incubated from time 0 to 8 h. At the time indicated, cells were processed

    Article Snippet: The MAPK inhibitors SB202190, PD98059 and SP600125 were from Calbiochem (San Diego, CA).

    Techniques: Incubation