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 sb202190
    SB 202190

    https://www.bioz.com/result/sb202190/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sb202190 - by Bioz Stars, 2021-03
    99/100 stars

    Images

    1) Product Images from "Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease"

    Article Title: Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20120707

    TTP regulates the production of IL23 in BMDCs. BMDCs from TTP +/+ or TTP −/− mice were incubated in medium alone or stimulated with 100 ng/ml LPS. (A) Supernatants were collected after 16 h and analyzed for IL23, IL12p70, and IL12/23p40 levels by ELISA. Mean ± SEM of six independent experiments is shown. (B) Total RNA was extracted and analyzed by real-time RT-PCR. One experiment representative of six is shown. (C and D) Spleen cells from TLR4 −/− mice were cultured in presence of supernatant from nonstimulated or LPS-stimulated DCs. When indicated, neutralizing antibodies for IL12/23p40, IL23p19, or control IgG (2 µg/ml) were added. After 3 d, supernatants were collected and analyzed for IL17A and IFN-γ levels by ELISA. Mean ± SEM of two experiments performed in triplicate (C) or triplicates from one representative experiment of three (D) is shown. (E) DCs from TTP +/+ or TTP −/− mice were stimulated with 100 ng/ml LPS. After 2 h of stimulation, 10 µg/ml actinomycin d and 1 µM SB202190 were added for the indicated time. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of five. (F and G) HEK-293 cells were cotransfected with 400 ng of the indicated reporter plasmid and 100 ng of the indicated expression vector. 24 h after transfection, culture medium was replaced by fresh medium, and 16 h later supernatant were collected and analyzed for IL2 levels by ELISA. Mean ± SEM from three experiments performed in triplicate is shown. (H) Representation of the 3′UTR of the murine IL23p19 mRNA. (I and J) Knockdown experiments were performed by nucleofection of 1 µM Caf1 or control (CTL) siRNA. 24 h later, cells were stimulated as indicated. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of two. *, P
    Figure Legend Snippet: TTP regulates the production of IL23 in BMDCs. BMDCs from TTP +/+ or TTP −/− mice were incubated in medium alone or stimulated with 100 ng/ml LPS. (A) Supernatants were collected after 16 h and analyzed for IL23, IL12p70, and IL12/23p40 levels by ELISA. Mean ± SEM of six independent experiments is shown. (B) Total RNA was extracted and analyzed by real-time RT-PCR. One experiment representative of six is shown. (C and D) Spleen cells from TLR4 −/− mice were cultured in presence of supernatant from nonstimulated or LPS-stimulated DCs. When indicated, neutralizing antibodies for IL12/23p40, IL23p19, or control IgG (2 µg/ml) were added. After 3 d, supernatants were collected and analyzed for IL17A and IFN-γ levels by ELISA. Mean ± SEM of two experiments performed in triplicate (C) or triplicates from one representative experiment of three (D) is shown. (E) DCs from TTP +/+ or TTP −/− mice were stimulated with 100 ng/ml LPS. After 2 h of stimulation, 10 µg/ml actinomycin d and 1 µM SB202190 were added for the indicated time. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of five. (F and G) HEK-293 cells were cotransfected with 400 ng of the indicated reporter plasmid and 100 ng of the indicated expression vector. 24 h after transfection, culture medium was replaced by fresh medium, and 16 h later supernatant were collected and analyzed for IL2 levels by ELISA. Mean ± SEM from three experiments performed in triplicate is shown. (H) Representation of the 3′UTR of the murine IL23p19 mRNA. (I and J) Knockdown experiments were performed by nucleofection of 1 µM Caf1 or control (CTL) siRNA. 24 h later, cells were stimulated as indicated. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of two. *, P

    Techniques Used: Mouse Assay, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Plasmid Preparation, Expressing, Transfection, CTL Assay

    2) Product Images from "Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization"

    Article Title: Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.22.7545-7557.2001

    Inhibitors of p38 MAPK block alkalinization induced by IL-7 or IL-3 withdrawal and enhance survival. (A) D1 cells were cultured in the presence or absence of IL-7 for 5 h and either not treated (None) or treated with 20 μM PD169316, 20 μM SB202190 (inhibitors of p38 MAPK), or 20 μM PD98059 (inhibitor of MEK1). (B) FL5.12A cells were incubated with or without IL-3 for 3 h and either not treated (None) or treated with 20 μM PD169316, 20 μM SB202190 (inhibitors of p38 MAPK), or 20 μM PD98059 (inhibitor of MEK1). Intracellular pH was measured as described in Materials and Methods. (C) The efficacy of the different MAPK inhibitors (20 μM) was tested in a p38 MAPK in vitro kinase assay using p38 MAPK immunoprecipiatated from anisomycintreated FL5.12A cells. Of the three p38 MAPK inhibitors assayed, PD169316 was the most effective in blocking p38 MAPK activity (shown at two concentrations, 20 and 10 μM), SB202190 was the second best, and SB203580 had minimal effect; the ERK inhibitor (PD98059) or the nonspecific inhibitor (SB202474) had no effect at the 20 μM dose. (D) GFP + cells or GFP + cells cotransfected with a DN-p38 MAPK were cultured in the absence of IL-3 for 48 h, and the percentage of cells with DNA fragmentation was measured by propidium iodide staining and flow cytometry analysis. Expression of the DN-p38 MAPK decreased DNA fragmentation by 20% during IL-3 withdrawal. GFP + ) and/or transfected with DN-p38 MAPK and evaluated 24 h later for pH changes after 1 h of anisomycin treatment by incorporation of BCECF as described in Materials and Methods.
    Figure Legend Snippet: Inhibitors of p38 MAPK block alkalinization induced by IL-7 or IL-3 withdrawal and enhance survival. (A) D1 cells were cultured in the presence or absence of IL-7 for 5 h and either not treated (None) or treated with 20 μM PD169316, 20 μM SB202190 (inhibitors of p38 MAPK), or 20 μM PD98059 (inhibitor of MEK1). (B) FL5.12A cells were incubated with or without IL-3 for 3 h and either not treated (None) or treated with 20 μM PD169316, 20 μM SB202190 (inhibitors of p38 MAPK), or 20 μM PD98059 (inhibitor of MEK1). Intracellular pH was measured as described in Materials and Methods. (C) The efficacy of the different MAPK inhibitors (20 μM) was tested in a p38 MAPK in vitro kinase assay using p38 MAPK immunoprecipiatated from anisomycintreated FL5.12A cells. Of the three p38 MAPK inhibitors assayed, PD169316 was the most effective in blocking p38 MAPK activity (shown at two concentrations, 20 and 10 μM), SB202190 was the second best, and SB203580 had minimal effect; the ERK inhibitor (PD98059) or the nonspecific inhibitor (SB202474) had no effect at the 20 μM dose. (D) GFP + cells or GFP + cells cotransfected with a DN-p38 MAPK were cultured in the absence of IL-3 for 48 h, and the percentage of cells with DNA fragmentation was measured by propidium iodide staining and flow cytometry analysis. Expression of the DN-p38 MAPK decreased DNA fragmentation by 20% during IL-3 withdrawal. GFP + ) and/or transfected with DN-p38 MAPK and evaluated 24 h later for pH changes after 1 h of anisomycin treatment by incorporation of BCECF as described in Materials and Methods.

    Techniques Used: Blocking Assay, Cell Culture, Incubation, In Vitro, Kinase Assay, Activity Assay, Staining, Flow Cytometry, Cytometry, Expressing, Transfection

    3) Product Images from "Acid ?-Glucosidase 1 Counteracts p38?-dependent Induction of Interleukin-6"

    Article Title: Acid ?-Glucosidase 1 Counteracts p38?-dependent Induction of Interleukin-6

    Journal:

    doi: 10.1074/jbc.M809500200

    Involvement of p38δ in the production of IL-6 in GBA1-silenced cells. A , MCF-7 cells were transfected with 5 n m SCR ( open column ) or GBA1 ( gray-filled column ) siRNAs for 48 h following pre-treatment with the indicated concentration of SB202190
    Figure Legend Snippet: Involvement of p38δ in the production of IL-6 in GBA1-silenced cells. A , MCF-7 cells were transfected with 5 n m SCR ( open column ) or GBA1 ( gray-filled column ) siRNAs for 48 h following pre-treatment with the indicated concentration of SB202190

    Techniques Used: Transfection, Concentration Assay

    4) Product Images from "Transforming Growth Factor ?1 (TGF-?1) Promotes Endothelial Cell Survival during In Vitro Angiogenesis via an Autocrine Mechanism Implicating TGF-? Signaling"

    Article Title: Transforming Growth Factor ?1 (TGF-?1) Promotes Endothelial Cell Survival during In Vitro Angiogenesis via an Autocrine Mechanism Implicating TGF-? Signaling

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.21.7218-7230.2001

    LY-294002 and PD-98059 prevent TGF-β1-induced tube formation and Ets-1 induction. (A) 1G11 endothelial cells immersed in collagen gels were cultured for 24 h in the presence of DMEM alone (basal) or 10 ng of TGF-β1/ml either alone or with 10 μM SB202190, 15 μM LY-294002 (Ly), 10 nM rapamycin (rapa), or 30 μM PD-98059 (PD). Cells were examined by phase-contrast microscopy. Magnification, ×128. (B) 1G11 cells grown in collagen gels for 24 h in the presence of 10 ng of TGF-β1/ml either alone (TGF-β1 lane −) or with 15 μM LY-294002 (lane +Ly) or 30 μM PD-98059 (lane +PD) or in the absence of TGF-β (B lane −) were lysed, and Ets-1 was detected by immunoblotting with a specific antibody. Identical amounts of protein were loaded on the gel. The decrease in p42 MAPK content in lanes +Ly and +PD reflects partial cell death. A representative Western blot of three different experiments is shown.
    Figure Legend Snippet: LY-294002 and PD-98059 prevent TGF-β1-induced tube formation and Ets-1 induction. (A) 1G11 endothelial cells immersed in collagen gels were cultured for 24 h in the presence of DMEM alone (basal) or 10 ng of TGF-β1/ml either alone or with 10 μM SB202190, 15 μM LY-294002 (Ly), 10 nM rapamycin (rapa), or 30 μM PD-98059 (PD). Cells were examined by phase-contrast microscopy. Magnification, ×128. (B) 1G11 cells grown in collagen gels for 24 h in the presence of 10 ng of TGF-β1/ml either alone (TGF-β1 lane −) or with 15 μM LY-294002 (lane +Ly) or 30 μM PD-98059 (lane +PD) or in the absence of TGF-β (B lane −) were lysed, and Ets-1 was detected by immunoblotting with a specific antibody. Identical amounts of protein were loaded on the gel. The decrease in p42 MAPK content in lanes +Ly and +PD reflects partial cell death. A representative Western blot of three different experiments is shown.

    Techniques Used: Cell Culture, Microscopy, Western Blot

    5) Product Images from "Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells"

    Article Title: Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfw114

    Effects of signal pathway inhibitors and resveratrol on inhibition of GJIC induced by MXC and VIN in WB-F344 cells. The cells were pretreated with MEK1/2 inhibitor, U0126 (20 µM, 30 min), p38 inhibitor SB202190 (25 µM, 30 min), phosphatidylcholine-specific phospholipase C inhibitor D609 (50 µM, 20 min), or with resveratrol (100 µM, 15 min), then exposed to MXC (20–25 µM, 10 min) or VIN (200–250 µM, 10 min). A, Representative images from SL-DT assay used for evaluation of GJIC. The effects of the chemicals were compared with the dye transfer in the negative control and expressed as FOC for both GJIC inhibitors, (B) MXC and (C) VIN. Data represent means ± SD from at least 3 independent experiments. Asterisks indicate values significantly different from the vehicle (Veh) control (Kruskal-Wallis ANOVA on Ranks followed by Dunn’s comparison method, P
    Figure Legend Snippet: Effects of signal pathway inhibitors and resveratrol on inhibition of GJIC induced by MXC and VIN in WB-F344 cells. The cells were pretreated with MEK1/2 inhibitor, U0126 (20 µM, 30 min), p38 inhibitor SB202190 (25 µM, 30 min), phosphatidylcholine-specific phospholipase C inhibitor D609 (50 µM, 20 min), or with resveratrol (100 µM, 15 min), then exposed to MXC (20–25 µM, 10 min) or VIN (200–250 µM, 10 min). A, Representative images from SL-DT assay used for evaluation of GJIC. The effects of the chemicals were compared with the dye transfer in the negative control and expressed as FOC for both GJIC inhibitors, (B) MXC and (C) VIN. Data represent means ± SD from at least 3 independent experiments. Asterisks indicate values significantly different from the vehicle (Veh) control (Kruskal-Wallis ANOVA on Ranks followed by Dunn’s comparison method, P

    Techniques Used: Inhibition, Western Blot, Negative Control

    6) Product Images from "Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins"

    Article Title: Knock Down of Heat Shock Protein 27 (HspB1) Induces Degradation of Several Putative Client Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029719

    Endogenous level of STAT2 is decreased in Hsp27 depleted cell line. A , Hsp27 depleted and control HeLa cells were collected, and samples were analyzed by immunoblotting using antibodies that recognize the different STAT polypeptides. Quantification of STA2 level was reported in a table. B , as in A ) but analysis of STAT2 level in MCF-7 depleted cells. C, Analysis of STAT3 phosphorylation. HeLa clones were treated with interleukin-6 (4 h; 125 ng/mL) or heat shock (60 min; 42°C and 44°C). Samples were collected and analyzed by immunobloting with STAT3, Phospho-STAT3, Hsp27 and actin antibodies. D , quantification of stat2 gene relative expression by qPCR. E , Effect of proteolytic inhibitors. HeLa cells were analyzed after being treated with MG132 or ALLN, as described in Fig. 3 . Level of STAT2 was revealed by immunobloting. F , HeLa cells were transiently transfected with an ISRE reporter gene construct [36] and treated for 6 h with 50 U/mL of interferon-α. They were also treated or not for 30 min with 10 µM of either SB203580 or SB202190 (p38MAPkinase and MK2 inhibitors, respectively) before exposure to interferon-α. Luciferase expression correlated with STAT2 transcriptional activity was quantified in both cell lines.
    Figure Legend Snippet: Endogenous level of STAT2 is decreased in Hsp27 depleted cell line. A , Hsp27 depleted and control HeLa cells were collected, and samples were analyzed by immunoblotting using antibodies that recognize the different STAT polypeptides. Quantification of STA2 level was reported in a table. B , as in A ) but analysis of STAT2 level in MCF-7 depleted cells. C, Analysis of STAT3 phosphorylation. HeLa clones were treated with interleukin-6 (4 h; 125 ng/mL) or heat shock (60 min; 42°C and 44°C). Samples were collected and analyzed by immunobloting with STAT3, Phospho-STAT3, Hsp27 and actin antibodies. D , quantification of stat2 gene relative expression by qPCR. E , Effect of proteolytic inhibitors. HeLa cells were analyzed after being treated with MG132 or ALLN, as described in Fig. 3 . Level of STAT2 was revealed by immunobloting. F , HeLa cells were transiently transfected with an ISRE reporter gene construct [36] and treated for 6 h with 50 U/mL of interferon-α. They were also treated or not for 30 min with 10 µM of either SB203580 or SB202190 (p38MAPkinase and MK2 inhibitors, respectively) before exposure to interferon-α. Luciferase expression correlated with STAT2 transcriptional activity was quantified in both cell lines.

    Techniques Used: Clone Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Construct, Luciferase, Activity Assay

    7) Product Images from "Negative regulation of human U6 snRNA promoter by p38 kinase through Oct-1"

    Article Title: Negative regulation of human U6 snRNA promoter by p38 kinase through Oct-1

    Journal: Gene

    doi: 10.1016/j.gene.2012.01.041

    SB202190 stimulated DSE-dependent U6 promoter activity. ( A ) Schematic illustration of the full length (pU6-264) and deletion mutants (pU6-200, -150, -100, -50) of U6 promoter (B) Validation of U6 promoter transcription by RNA Pol III. pU6-264 or pCMV-luc
    Figure Legend Snippet: SB202190 stimulated DSE-dependent U6 promoter activity. ( A ) Schematic illustration of the full length (pU6-264) and deletion mutants (pU6-200, -150, -100, -50) of U6 promoter (B) Validation of U6 promoter transcription by RNA Pol III. pU6-264 or pCMV-luc

    Techniques Used: Activity Assay

    8) Product Images from "Hepatitis B Virus X Protein Enhances Cisplatin-Induced Hepatotoxicity via a Mechanism Involving Degradation of Mcl-1 ▿Hepatitis B Virus X Protein Enhances Cisplatin-Induced Hepatotoxicity via a Mechanism Involving Degradation of Mcl-1 ▿ †"

    Article Title: Hepatitis B Virus X Protein Enhances Cisplatin-Induced Hepatotoxicity via a Mechanism Involving Degradation of Mcl-1 ▿Hepatitis B Virus X Protein Enhances Cisplatin-Induced Hepatotoxicity via a Mechanism Involving Degradation of Mcl-1 ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01841-10

    Caspase inhibitors abrogate HBx-enhanced Mcl-1 degradation. (A and B) Cells were pretreated with DMSO, the JNK-specific inhibitor SP600125 (10 μM) (A), or the p38-specific inhibitor SB202190 (10 μM) (B), followed by exposure to cisplatin
    Figure Legend Snippet: Caspase inhibitors abrogate HBx-enhanced Mcl-1 degradation. (A and B) Cells were pretreated with DMSO, the JNK-specific inhibitor SP600125 (10 μM) (A), or the p38-specific inhibitor SB202190 (10 μM) (B), followed by exposure to cisplatin

    Techniques Used:

    9) Product Images from "p38 MAPK-inhibited dendritic cells induce superior antitumor immune responses and overcome regulatory T cell-mediated immunosuppression"

    Article Title: p38 MAPK-inhibited dendritic cells induce superior antitumor immune responses and overcome regulatory T cell-mediated immunosuppression

    Journal: Nature communications

    doi: 10.1038/ncomms5229

    Downregulation of PPARγ expression/activity in mSBDCs contributes to the p50-mediated increased expression of surface OX40L. ( a ) Western blot analysis of the PPARγ, pC/EBPβ and C/EBPβ in mDCs and mSBDCs treated with or without p38 inhibitor SB202190, PPARγ inhibitor GW9662, and/or PPARγ activator RGZ. ( b ) The protein level of PPARγ, pC/EBPβ and C/EBPβ in mDCs treated with C/EBPβ-specific siRNA. ( c ) RT-PCR analysis of Ap2 mRNA expression in mDCs treated with or without p38 inhibitor SB202190, PPARγ inhibitor GW9662, PPARγ activator RGZ, and/or p38 activator anisomycin. Data shown (n = 3) were normalized to the Gapdh gene. Error bars represent s.d. ( d ) Western blot analysis of the cytoplasmic and nuclear localization of p50 molecule in treated mDCs and mSBDCs. ( e ) Coimmunoprecipitation of endogenous p50 and PPARγ. mDCs and mSBDCs lysates were immunoprecipitated with PPARγ antibody or isotype control IgG and immunoblotted with p50 antibody. ( f ) Surface expression of OX40L on treated mDCs and mSBDCs generated from wild-type and p50 −/− mice analyzed by FACS. Numbers showed the percentage of OX40L-positive DCs gated on CD11c + cells. ( g ) Summarized data (n = 4) from ( f ). Error bars represent s.d. Representative data from one of two performed experiments are shown. P values were calculated with Student’s t -test.
    Figure Legend Snippet: Downregulation of PPARγ expression/activity in mSBDCs contributes to the p50-mediated increased expression of surface OX40L. ( a ) Western blot analysis of the PPARγ, pC/EBPβ and C/EBPβ in mDCs and mSBDCs treated with or without p38 inhibitor SB202190, PPARγ inhibitor GW9662, and/or PPARγ activator RGZ. ( b ) The protein level of PPARγ, pC/EBPβ and C/EBPβ in mDCs treated with C/EBPβ-specific siRNA. ( c ) RT-PCR analysis of Ap2 mRNA expression in mDCs treated with or without p38 inhibitor SB202190, PPARγ inhibitor GW9662, PPARγ activator RGZ, and/or p38 activator anisomycin. Data shown (n = 3) were normalized to the Gapdh gene. Error bars represent s.d. ( d ) Western blot analysis of the cytoplasmic and nuclear localization of p50 molecule in treated mDCs and mSBDCs. ( e ) Coimmunoprecipitation of endogenous p50 and PPARγ. mDCs and mSBDCs lysates were immunoprecipitated with PPARγ antibody or isotype control IgG and immunoblotted with p50 antibody. ( f ) Surface expression of OX40L on treated mDCs and mSBDCs generated from wild-type and p50 −/− mice analyzed by FACS. Numbers showed the percentage of OX40L-positive DCs gated on CD11c + cells. ( g ) Summarized data (n = 4) from ( f ). Error bars represent s.d. Representative data from one of two performed experiments are shown. P values were calculated with Student’s t -test.

    Techniques Used: Expressing, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Generated, Mouse Assay, FACS

    The role of p38 MAPK signaling in the regulation of DC immunogenicity. p38 MAPK phosphorylates C/EBPβ and pC/EBPβ induces the production of PPARγ, PPARγ can be activated by its ligand or agonist, which can then repress the transactivation of p50 by physical interaction with p50 to prevent its binding to the response elements such as Ox40l promoter. p38 MAPK inhibitor, such as SB202190, could abrogate this self-limitation break and result in a robust p50 nuclear translocation. p50, after forming transcriptionally active complexes with RelB, induces the strikingly increased surface OX40L expression, which is crucial to activation of Teff and inhibition of Treg.
    Figure Legend Snippet: The role of p38 MAPK signaling in the regulation of DC immunogenicity. p38 MAPK phosphorylates C/EBPβ and pC/EBPβ induces the production of PPARγ, PPARγ can be activated by its ligand or agonist, which can then repress the transactivation of p50 by physical interaction with p50 to prevent its binding to the response elements such as Ox40l promoter. p38 MAPK inhibitor, such as SB202190, could abrogate this self-limitation break and result in a robust p50 nuclear translocation. p50, after forming transcriptionally active complexes with RelB, induces the strikingly increased surface OX40L expression, which is crucial to activation of Teff and inhibition of Treg.

    Techniques Used: Binding Assay, Translocation Assay, Expressing, Activation Assay, Inhibition

    10) Product Images from "Metformin Promotes Apoptosis but Suppresses Autophagy in Glucose-Deprived H4IIE Hepatocellular Carcinoma Cells"

    Article Title: Metformin Promotes Apoptosis but Suppresses Autophagy in Glucose-Deprived H4IIE Hepatocellular Carcinoma Cells

    Journal: Diabetes & Metabolism Journal

    doi: 10.4093/dmj.2015.39.6.518

    Inhibition of p38 mitogen-activated protein kinase (p38MAPK) as well as AMP-activated protein kinase protected cells from apoptosis induced by metformin (Met). H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with various inhibitors against signaling proteins (20 µM compound C [CC], 50 µM SB202190, 50 µM SP600125, 10 µM U0126, 100 nM rapamycin, and 100 nM wortmannin) for 30 minutes. Cells were further treated 1 mM Met for 24 hours. After the treatments, the protein levels of cleaved caspase-3, beclin-1, light chain 3B (LC3B), and actin were detected using Western blotting analyses (A, C, D), and cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay (B, E). Each bar represents the mean±standard error ( n =3) (B, E). Rapa, rapamycin; SB, SB202190; SP, SP600125; U, U0126; Wt, wortmannin. a P
    Figure Legend Snippet: Inhibition of p38 mitogen-activated protein kinase (p38MAPK) as well as AMP-activated protein kinase protected cells from apoptosis induced by metformin (Met). H4IIE cells were pre-incubated in serum-free Dulbecco's minimal essential medium (DMEM, 1 g/L glucose) for 24 hours and then pretreated with various inhibitors against signaling proteins (20 µM compound C [CC], 50 µM SB202190, 50 µM SP600125, 10 µM U0126, 100 nM rapamycin, and 100 nM wortmannin) for 30 minutes. Cells were further treated 1 mM Met for 24 hours. After the treatments, the protein levels of cleaved caspase-3, beclin-1, light chain 3B (LC3B), and actin were detected using Western blotting analyses (A, C, D), and cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay (B, E). Each bar represents the mean±standard error ( n =3) (B, E). Rapa, rapamycin; SB, SB202190; SP, SP600125; U, U0126; Wt, wortmannin. a P

    Techniques Used: Inhibition, Incubation, Western Blot, MTT Assay

    11) Product Images from "Involvement of p38 MAPK in the Drug Resistance of Refractory Epilepsy Through the Regulation Multidrug Resistance-Associated Protein 1"

    Article Title: Involvement of p38 MAPK in the Drug Resistance of Refractory Epilepsy Through the Regulation Multidrug Resistance-Associated Protein 1

    Journal: Neurochemical Research

    doi: 10.1007/s11064-015-1617-y

    a MRP1 and p38 MAPK expression in the rat brain (fluorescence microscope 400×). a Cortex of the control group; b Cortex of the epilepsy group; c Cortex of the SB202190 group; d Hippocampus CA1 region of the control group; e Hippocampus CA1 region of the epilepsy group; f Hippocampus CA1 region of the SB202190 group. b Quantitative analysis of immunofluorescence. MRP1-positive and p38 MAPK-positive cells were counted and at least 3 slices from each brain were measured. ** p
    Figure Legend Snippet: a MRP1 and p38 MAPK expression in the rat brain (fluorescence microscope 400×). a Cortex of the control group; b Cortex of the epilepsy group; c Cortex of the SB202190 group; d Hippocampus CA1 region of the control group; e Hippocampus CA1 region of the epilepsy group; f Hippocampus CA1 region of the SB202190 group. b Quantitative analysis of immunofluorescence. MRP1-positive and p38 MAPK-positive cells were counted and at least 3 slices from each brain were measured. ** p

    Techniques Used: Expressing, Fluorescence, Microscopy, Immunofluorescence

    12) Product Images from "The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines"

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0147-4

    SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.
    Figure Legend Snippet: SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.

    Techniques Used: Western Blot, Software

    Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.
    Figure Legend Snippet: Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.

    Techniques Used: MTT Assay

    Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p
    Figure Legend Snippet: Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p

    Techniques Used: Expressing, Cell Culture

    HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p
    Figure Legend Snippet: HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p

    Techniques Used: Activity Assay, Stable Transfection, Transfection, Luciferase

    13) Product Images from "The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines"

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0147-4

    SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.
    Figure Legend Snippet: SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.

    Techniques Used: Western Blot, Software

    Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.
    Figure Legend Snippet: Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.

    Techniques Used: MTT Assay

    Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p
    Figure Legend Snippet: Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p

    Techniques Used: Expressing, Cell Culture

    HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p
    Figure Legend Snippet: HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p

    Techniques Used: Activity Assay, Stable Transfection, Transfection, Luciferase

    14) Product Images from "The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines"

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0147-4

    SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.
    Figure Legend Snippet: SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.

    Techniques Used: Western Blot, Software

    Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.
    Figure Legend Snippet: Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.

    Techniques Used: MTT Assay

    Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p
    Figure Legend Snippet: Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p

    Techniques Used: Expressing, Cell Culture

    HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p
    Figure Legend Snippet: HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p

    Techniques Used: Activity Assay, Stable Transfection, Transfection, Luciferase

    15) Product Images from "The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines"

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0147-4

    SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.
    Figure Legend Snippet: SB202190 enhances cleavage of PARP when combined with glucose analogs. Western blot analysis of whole cell lysates from cells treated for 24 hours with 2-DG or D-allose alone or in combination with SB202190. Lysates were probed for cleaved and total PARP. Tubulin served as a loading control for protein. Cleaved PARP band density was quantified using ImageJ software with normalization to α-Tubulin. One representative experiment is shown, n = 3.

    Techniques Used: Western Blot, Software

    Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.
    Figure Legend Snippet: Effect of dosing regimens on the proliferation of pancreatic and ovarian cell lines following 48 hours of treatment. Cells were treated with increasing doses of 2-DG or D-allose alone or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines) and/or SB202190. Cell proliferation was measured using the MTT assay. The ratio of 2-DG or D-allose to the platinum analogs was 2000:1 for all doses, respectively. One representative experiment is shown, n = 6.

    Techniques Used: MTT Assay

    Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p
    Figure Legend Snippet: Lactate production and HIF-1α expression in pancreatic cancer cell lines. A) . Effect of 2-DG and D-allose alone or in combination with SB202190 following 24 hours of treatment on lactate production measured in cell culture media or in cells. Values are normalized to total cell protein. One representative experiment is shown, n = 3. *p

    Techniques Used: Expressing, Cell Culture

    HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p
    Figure Legend Snippet: HIF-1α transcriptional activity is modulated by SB202190. A) . MIA PaCa-2 cells were stably transfected with a HRE-luciferase reporter and treated for 3 24, and 48 hours with 2-DG or D-allose alone and in combination with SB202190. Luciferase activity was normalized to total protein. One representative experiment is shown, n = 3. *Indicates a p

    Techniques Used: Activity Assay, Stable Transfection, Transfection, Luciferase

    16) Product Images from "Antimicrobial Peptide TP4 Induces ROS-Mediated Necrosis by Triggering Mitochondrial Dysfunction in Wild-Type and Mutant p53 Glioblastoma Cells"

    Article Title: Antimicrobial Peptide TP4 Induces ROS-Mediated Necrosis by Triggering Mitochondrial Dysfunction in Wild-Type and Mutant p53 Glioblastoma Cells

    Journal: Cancers

    doi: 10.3390/cancers11020171

    p38-mediated protective effect limits TP4-induced cytotoxicity. ( A ) Cells were treated with TP4 (20 μg/mL) for different times. Cell lysates were collected and immunoblotted with phospho-p38 (P-p38) and p38 antibodies. Bottom panel: quantification of P-p38. Cells were preincubated with a specific p38 inhibitor, SB202190 (20 μM) for 1 h, followed by TP4 (20 μg/mL) treatment for 24 h. Cell number was determined by the trypan exclusion assay ( B ), and the percentage of PI + cells was assessed using flow cytometry ( C ) Vehicle: 0.5% DMSO. ( D ) Cells were treated as described for ( B ) and ( C ). Supernatants were immunoblotted with cyclophilin A antibody. Cell lysates were immunoblotted with β-actin antibody. Bottom panel: quantification of cyclophilin A. Veh (vehicle): 0.5% DMSO; SB: SB202190; T+SB: TP4+SB202190. ( E ) Cells were preincubated with SB202190 (20 μM) for 1 h, followed by TP4 (20 μg/mL) treatment for 1 h. ROS were labeled using DHE (20 μM) and DCF-DA (10 μM). Fluorescence intensity was assessed using flow cytometry. Cells were preincubated with a specific p38 inhibitor, SB202190 (20 μM) for 1 h, followed by TP4 (20 μg/mL) treatment for 1 h. The fluorescence intensity of TMRE ( F ) and MitoTracker Red CMXRos ( G ) was assessed using flow cytometry. Cells were preincubated with ROS scavenger NAC (5 mM) ( H ) or MitoTEMPO (10 μM) ( I ) for 1 h, followed by TP4 (20 μg/mL) treatment for 0.5 h. Cell lysates were collected and immunoblotted with P-p38 (phospho-p38) and p38 antibodies. Bottom panel: quantification of P-p38. Con: control; Veh: vehicle (0.5% DMSO); Mito: MitoTEMPO; T + Mito: TP4 + MitoTEMPO; T+NAC: TP4 + NAC. Western blotting experiments were performed at least three times with similar results. Band intensities were quantified with Image J software. * p
    Figure Legend Snippet: p38-mediated protective effect limits TP4-induced cytotoxicity. ( A ) Cells were treated with TP4 (20 μg/mL) for different times. Cell lysates were collected and immunoblotted with phospho-p38 (P-p38) and p38 antibodies. Bottom panel: quantification of P-p38. Cells were preincubated with a specific p38 inhibitor, SB202190 (20 μM) for 1 h, followed by TP4 (20 μg/mL) treatment for 24 h. Cell number was determined by the trypan exclusion assay ( B ), and the percentage of PI + cells was assessed using flow cytometry ( C ) Vehicle: 0.5% DMSO. ( D ) Cells were treated as described for ( B ) and ( C ). Supernatants were immunoblotted with cyclophilin A antibody. Cell lysates were immunoblotted with β-actin antibody. Bottom panel: quantification of cyclophilin A. Veh (vehicle): 0.5% DMSO; SB: SB202190; T+SB: TP4+SB202190. ( E ) Cells were preincubated with SB202190 (20 μM) for 1 h, followed by TP4 (20 μg/mL) treatment for 1 h. ROS were labeled using DHE (20 μM) and DCF-DA (10 μM). Fluorescence intensity was assessed using flow cytometry. Cells were preincubated with a specific p38 inhibitor, SB202190 (20 μM) for 1 h, followed by TP4 (20 μg/mL) treatment for 1 h. The fluorescence intensity of TMRE ( F ) and MitoTracker Red CMXRos ( G ) was assessed using flow cytometry. Cells were preincubated with ROS scavenger NAC (5 mM) ( H ) or MitoTEMPO (10 μM) ( I ) for 1 h, followed by TP4 (20 μg/mL) treatment for 0.5 h. Cell lysates were collected and immunoblotted with P-p38 (phospho-p38) and p38 antibodies. Bottom panel: quantification of P-p38. Con: control; Veh: vehicle (0.5% DMSO); Mito: MitoTEMPO; T + Mito: TP4 + MitoTEMPO; T+NAC: TP4 + NAC. Western blotting experiments were performed at least three times with similar results. Band intensities were quantified with Image J software. * p

    Techniques Used: Exclusion Assay, Flow Cytometry, Cytometry, Labeling, Fluorescence, Western Blot, Software

    ROS are crucial for TP4-induced DNA damage. ( A ) Cells were treated with TP4 (20 μg/mL) for different times. Cell lysates were collected and immunoblotted with P-H2A.X (phospho-H2A.X) and β-actin antibodies. Bottom panel: quantification of P-H2A.X. ( B ) Comet assay results of TP4 exposure. Cells were treated with or without TP4 (20 μg/mL) for 24 h. Bottom panel: percentage of DNA tail was measured in 100 cells. Cells were preincubated with ROS scavenger NAC (5 mM), ( C ), MitoTEMPO (10 μM) ( D ) or SB202190 (20 μM) ( E ) for 1 h, followed by TP4 (20 μg/mL) treatment for 24 h. Cell lysates were collected and immunoblotted with P-H2A.X and β-actin antibodies. Bottom panel: quantification of P-H2A.X. Con: control; Veh: vehicle (0.5% DMSO); SB: SB202190; T + SB: TP4 + SB202190; Mito: MitoTEMPO; T + Mito: TP4 + MitoTEMPO; T + NAC: TP4 + NAC. Western blotting experiments were performed at least three times with similar results. Band intensities were quantified with Image J software. * p
    Figure Legend Snippet: ROS are crucial for TP4-induced DNA damage. ( A ) Cells were treated with TP4 (20 μg/mL) for different times. Cell lysates were collected and immunoblotted with P-H2A.X (phospho-H2A.X) and β-actin antibodies. Bottom panel: quantification of P-H2A.X. ( B ) Comet assay results of TP4 exposure. Cells were treated with or without TP4 (20 μg/mL) for 24 h. Bottom panel: percentage of DNA tail was measured in 100 cells. Cells were preincubated with ROS scavenger NAC (5 mM), ( C ), MitoTEMPO (10 μM) ( D ) or SB202190 (20 μM) ( E ) for 1 h, followed by TP4 (20 μg/mL) treatment for 24 h. Cell lysates were collected and immunoblotted with P-H2A.X and β-actin antibodies. Bottom panel: quantification of P-H2A.X. Con: control; Veh: vehicle (0.5% DMSO); SB: SB202190; T + SB: TP4 + SB202190; Mito: MitoTEMPO; T + Mito: TP4 + MitoTEMPO; T + NAC: TP4 + NAC. Western blotting experiments were performed at least three times with similar results. Band intensities were quantified with Image J software. * p

    Techniques Used: Single Cell Gel Electrophoresis, Western Blot, Software

    17) Product Images from "Monosodium urate crystal-induced pro-interleukin-1β production is post-transcriptionally regulated via the p38 signaling pathway in human monocytes"

    Article Title: Monosodium urate crystal-induced pro-interleukin-1β production is post-transcriptionally regulated via the p38 signaling pathway in human monocytes

    Journal: Scientific Reports

    doi: 10.1038/srep34533

    MSU crystal-induced production of pro-IL-1β is controlled by p38 MAP kinase signaling pathways. (a ) Human primary monocytes were stimulated with PBS or MSU crystals (400 μg/ml) for the indicated times. Phosphorylation of NF-κB, ERK, p38, mTOR, and 4E-BP1 was assessed in monocyte lysates by immunoblot analysis with phospho-specific and total protein-specific antibodies. ( b ) Monocytes were stimulated with MSU crystals in the presence of inhibitors: 50 nM rapamycin (Ra: allosteric mTOR inhibitor), 50 nM Torin1 (Tor: selective mTOR C1/2 inhibitor), 20 μM PD98059 (PD: a potent inhibitor of MEK1), 10 μM FR180204 (FR: selective ERK1/2 inhibitor), 5 μM SB202190 (p38α and β isoform selective inhibitor), and DMSO (D: vehicle control). Monocytes were pretreated with these inhibitors for 30 min prior to stimulation with MSU crystals for 60 min. Cell lysates were analyzed by immunoblot for the effect of the inhibitors on pro-IL-1β synthesis. Intensities between upper and lower blots were normalized to vehicle control (1 st lane). ( c ) The effect of the inhibitors on pro-IL-1β synthesis is presented as fold change compared with vehicle control (1 st lane). Values were normalized to β-actin. The scatter plots show the mean from three to four independent experiments with three to four different donors. N.S. indicates not significant, * p
    Figure Legend Snippet: MSU crystal-induced production of pro-IL-1β is controlled by p38 MAP kinase signaling pathways. (a ) Human primary monocytes were stimulated with PBS or MSU crystals (400 μg/ml) for the indicated times. Phosphorylation of NF-κB, ERK, p38, mTOR, and 4E-BP1 was assessed in monocyte lysates by immunoblot analysis with phospho-specific and total protein-specific antibodies. ( b ) Monocytes were stimulated with MSU crystals in the presence of inhibitors: 50 nM rapamycin (Ra: allosteric mTOR inhibitor), 50 nM Torin1 (Tor: selective mTOR C1/2 inhibitor), 20 μM PD98059 (PD: a potent inhibitor of MEK1), 10 μM FR180204 (FR: selective ERK1/2 inhibitor), 5 μM SB202190 (p38α and β isoform selective inhibitor), and DMSO (D: vehicle control). Monocytes were pretreated with these inhibitors for 30 min prior to stimulation with MSU crystals for 60 min. Cell lysates were analyzed by immunoblot for the effect of the inhibitors on pro-IL-1β synthesis. Intensities between upper and lower blots were normalized to vehicle control (1 st lane). ( c ) The effect of the inhibitors on pro-IL-1β synthesis is presented as fold change compared with vehicle control (1 st lane). Values were normalized to β-actin. The scatter plots show the mean from three to four independent experiments with three to four different donors. N.S. indicates not significant, * p

    Techniques Used:

    Inhibition of Mnk1, a p38 MAPK substrate, suppresses MSU crystal-stimulated pro-IL-1β and global protein synthesis. ( a ) Human primary monocytes were stimulated with PBS or MSU crystals (400 μg/ml) for the indicated times. Phosphorylation of MnK1 and eIF4E were assessed in monocyte lysates by immunoblot analysis with phospho-specific and total protein-specific antibodies. Data is representative of two independent experiments with two different donors. ( b ) Human monocytes were pre-treated for 30 min with the indicated concentrations of CGP57380 (Mnk1 inhibitor), SB202190 (5 μM), or DMSO (vehicle) prior to stimulation with MSU crystals (400 μg/ml) for 60 min. Cell lysates were analyzed by immunoblot for the effect of the inhibitors on pro-IL-1β synthesis. The values between upper and lower blots were normalized to vehicle control (1 st lane). Data is representative of two independent experiments with two different donors. ( c ) Biotin-conjugated newly-synthesized proteins (Input) were collected using streptavidin-agarose (Pull-down) and the indicated proteins were examined by immunoblot analysis using streptavidin-HRP (upper panel) or anti-IL-1β antibody (lower panel). Data is representative of three independent experiments with three different donors. ( d ) The inhibitory effect of the CGP57380 on the synthesis of nascent global proteins and pro-IL-1β protein is presented as fold change compared with vehicle control (1 st lane). The values of global and pro-IL-1β proteins were normalized to β-actin (Input) and total biotinylated protein (Pull-down), respectively. The scatter plots show the mean of three independent experiments with three different donors. N.S. indicates not significant, * p
    Figure Legend Snippet: Inhibition of Mnk1, a p38 MAPK substrate, suppresses MSU crystal-stimulated pro-IL-1β and global protein synthesis. ( a ) Human primary monocytes were stimulated with PBS or MSU crystals (400 μg/ml) for the indicated times. Phosphorylation of MnK1 and eIF4E were assessed in monocyte lysates by immunoblot analysis with phospho-specific and total protein-specific antibodies. Data is representative of two independent experiments with two different donors. ( b ) Human monocytes were pre-treated for 30 min with the indicated concentrations of CGP57380 (Mnk1 inhibitor), SB202190 (5 μM), or DMSO (vehicle) prior to stimulation with MSU crystals (400 μg/ml) for 60 min. Cell lysates were analyzed by immunoblot for the effect of the inhibitors on pro-IL-1β synthesis. The values between upper and lower blots were normalized to vehicle control (1 st lane). Data is representative of two independent experiments with two different donors. ( c ) Biotin-conjugated newly-synthesized proteins (Input) were collected using streptavidin-agarose (Pull-down) and the indicated proteins were examined by immunoblot analysis using streptavidin-HRP (upper panel) or anti-IL-1β antibody (lower panel). Data is representative of three independent experiments with three different donors. ( d ) The inhibitory effect of the CGP57380 on the synthesis of nascent global proteins and pro-IL-1β protein is presented as fold change compared with vehicle control (1 st lane). The values of global and pro-IL-1β proteins were normalized to β-actin (Input) and total biotinylated protein (Pull-down), respectively. The scatter plots show the mean of three independent experiments with three different donors. N.S. indicates not significant, * p

    Techniques Used: Inhibition, Synthesized

    MSU crystal-stimulated pro-IL-1β protein synthesis is dependent on p38 MAPK, but not on mTOR. Click-iT ® Labeling and pull-down assay of biotinylated proteins was performed as previously described in Fig. 2 . Human monocytes were stimulated with MSU crystals for 60 min after 30 min pretreatment with inhibitors: Rapamycin (50 nM), Torin 1 (50 nM), PD98059 (20 μM), FR180204 (10 μM), SB202190 (5 μM), and DMSO (vehicle control). ( a ) Coomassie blue staining was used to analyze the amount of total proteins in each Click-iT-reacted sample after polyacrylamide gel electrophoresis. ( b ) Biotin-conjugated newly-synthesized proteins (Input) were collected by streptavidin-agarose (Pull-down) and the indicated proteins were examined by immunoblot analysis using streptavidin-HRP (upper panel) or anti-IL-1β antibody (lower panel). ( c , d ) The effect of the inhibitors on the synthesis of nascent global proteins ( c ) and pro-IL-1β protein ( d ) is presented as fold change compared with vehicle control (1 st lane). Global protein and pro-IL-1β protein were normalized to β-actin (Input) and total biotinylated protein (Pull-down), respectively. Data is representative of two independent experiments with two different donors.
    Figure Legend Snippet: MSU crystal-stimulated pro-IL-1β protein synthesis is dependent on p38 MAPK, but not on mTOR. Click-iT ® Labeling and pull-down assay of biotinylated proteins was performed as previously described in Fig. 2 . Human monocytes were stimulated with MSU crystals for 60 min after 30 min pretreatment with inhibitors: Rapamycin (50 nM), Torin 1 (50 nM), PD98059 (20 μM), FR180204 (10 μM), SB202190 (5 μM), and DMSO (vehicle control). ( a ) Coomassie blue staining was used to analyze the amount of total proteins in each Click-iT-reacted sample after polyacrylamide gel electrophoresis. ( b ) Biotin-conjugated newly-synthesized proteins (Input) were collected by streptavidin-agarose (Pull-down) and the indicated proteins were examined by immunoblot analysis using streptavidin-HRP (upper panel) or anti-IL-1β antibody (lower panel). ( c , d ) The effect of the inhibitors on the synthesis of nascent global proteins ( c ) and pro-IL-1β protein ( d ) is presented as fold change compared with vehicle control (1 st lane). Global protein and pro-IL-1β protein were normalized to β-actin (Input) and total biotinylated protein (Pull-down), respectively. Data is representative of two independent experiments with two different donors.

    Techniques Used: Labeling, Pull Down Assay, Staining, Polyacrylamide Gel Electrophoresis, Synthesized

    MSU crystals increase pro-IL-1β mRNA stability, which is controlled by p38 MAPK signaling pathway via MK2. ( a ) Human primary monocytes were stimulated with PBS or MSU crystals (400 μg/ml) for the indicated times. Phosphorylation of MK2 was assessed in monocyte lysates by immunoblot analysis with phospho-specific and total protein-specific antibodies. ( b ) Cells were treated with Act D (2.5 μg/ml) prior to stimulation with MSU crystals (400 μg/ml) or PBS for the indicated times. RNA was isolated from the monocytes and the level of pro-IL-1β transcription was determined using SYBR green qPCR. The level of the pro-IL-1β mRNA before treatment with Act D and MSU crystals was considered as 100%. Human GAPDH was used for normalization. The scatter plots show the mean in triplicate from three ndependent experiments with three different donors. ( c ) The effect of inhibitors including Rapamycin (50 nM), FR180204 (10 μM), CGP57380 (Mnk1 inhibitor, 20 μM), MK25 (MK2 inhibitor, 10 μM), and SB202190 (5 μM) on pro-IL-1β mRNA stability is presented as the % change compared with vehicle control (1 st lane). The scatter plots show the mean in triplicate from three to four independent experiments with three to four different donors. ( d ) Human monocytes were pre-treated for 30 min with the indicated concentrations of MK25 (MK2 inhibitor), CGP57380 (Mnk1 inhibitor), SB202190, or DMSO (vehicle) prior to stimulation with MSU crystals (400 μg/ml) for 60 min. Cell lysates were analyzed by immunoblot for pro-IL-1β production. The graph shows the mean ± SEM in triplicate from three independent experiments with three different donors. ( e ) Biotin-conjugated newly-synthesized proteins (Input) were collected using streptavidin-agarose (Pull-down) and the indicated proteins were examined by immunoblot analysis using streptavidin-HRP (upper panel) or anti-IL-1β antibody (lower panel). Blots are two independent experiments with two different donors. * p
    Figure Legend Snippet: MSU crystals increase pro-IL-1β mRNA stability, which is controlled by p38 MAPK signaling pathway via MK2. ( a ) Human primary monocytes were stimulated with PBS or MSU crystals (400 μg/ml) for the indicated times. Phosphorylation of MK2 was assessed in monocyte lysates by immunoblot analysis with phospho-specific and total protein-specific antibodies. ( b ) Cells were treated with Act D (2.5 μg/ml) prior to stimulation with MSU crystals (400 μg/ml) or PBS for the indicated times. RNA was isolated from the monocytes and the level of pro-IL-1β transcription was determined using SYBR green qPCR. The level of the pro-IL-1β mRNA before treatment with Act D and MSU crystals was considered as 100%. Human GAPDH was used for normalization. The scatter plots show the mean in triplicate from three ndependent experiments with three different donors. ( c ) The effect of inhibitors including Rapamycin (50 nM), FR180204 (10 μM), CGP57380 (Mnk1 inhibitor, 20 μM), MK25 (MK2 inhibitor, 10 μM), and SB202190 (5 μM) on pro-IL-1β mRNA stability is presented as the % change compared with vehicle control (1 st lane). The scatter plots show the mean in triplicate from three to four independent experiments with three to four different donors. ( d ) Human monocytes were pre-treated for 30 min with the indicated concentrations of MK25 (MK2 inhibitor), CGP57380 (Mnk1 inhibitor), SB202190, or DMSO (vehicle) prior to stimulation with MSU crystals (400 μg/ml) for 60 min. Cell lysates were analyzed by immunoblot for pro-IL-1β production. The graph shows the mean ± SEM in triplicate from three independent experiments with three different donors. ( e ) Biotin-conjugated newly-synthesized proteins (Input) were collected using streptavidin-agarose (Pull-down) and the indicated proteins were examined by immunoblot analysis using streptavidin-HRP (upper panel) or anti-IL-1β antibody (lower panel). Blots are two independent experiments with two different donors. * p

    Techniques Used: Activated Clotting Time Assay, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Synthesized

    18) Product Images from "Iron sensitizes keratinocytes and fibroblasts to UVA-mediated matrix metalloproteinase-1 through TNF-? and ERK activation"

    Article Title: Iron sensitizes keratinocytes and fibroblasts to UVA-mediated matrix metalloproteinase-1 through TNF-? and ERK activation

    Journal: Experimental dermatology

    doi: 10.1111/j.1600-0625.2010.01152.x

    Participation of ERK pathway in iron- and UVA-mediated metalloproteinase-1 activities. (a) Fibroblasts grown under Post-condition were pretreated with different kinase inhibitors for 1 h before UVA exposure. DMSO was used as control, PD98059 (final concentration 10 μM) as ERK inhibitor, U0126 (20 μM) as MEK1 inhibitor, SP600126 (10 μM) as JNK inhibitor, SB202190 (10 μM) as P38 inhibitor, Wortmannin (0.5 μM) as PI-3K inhibitor. (b) Phosphorylations of ERK, P38 and JNK were measured in fibroblasts grown under Pre- and Post-conditions and post UVA exposure. *Significantly different from their respective controls ( P
    Figure Legend Snippet: Participation of ERK pathway in iron- and UVA-mediated metalloproteinase-1 activities. (a) Fibroblasts grown under Post-condition were pretreated with different kinase inhibitors for 1 h before UVA exposure. DMSO was used as control, PD98059 (final concentration 10 μM) as ERK inhibitor, U0126 (20 μM) as MEK1 inhibitor, SP600126 (10 μM) as JNK inhibitor, SB202190 (10 μM) as P38 inhibitor, Wortmannin (0.5 μM) as PI-3K inhibitor. (b) Phosphorylations of ERK, P38 and JNK were measured in fibroblasts grown under Pre- and Post-conditions and post UVA exposure. *Significantly different from their respective controls ( P

    Techniques Used: Concentration Assay

    Related Articles

    Inhibition:

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines
    Article Snippet: .. SB202190 enhances 2-DG and D-allose mediated PARP cleavage To investigate if p38 MAPK inhibition sensitizes cancer cells to apoptosis we pre-treated MIA PaCa-2 cells with SB202190 for 2 hours and then added 10 mM 2-DG or 10 mM D-allose (with no washout of SB202190) for 24 hours. .. Western blot analysis of whole cell lysates was performed and the blots probed for cleaved and total PARP (Figure ).

    other:

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines
    Article Snippet: SB202190 further reduced lactate accumulation in combination with both glucose analogs in the MIA PaCA-2 cell line, but had mixed effects in the BxPC-3 and AsPC-1 cells.

    Article Title: Acid ?-Glucosidase 1 Counteracts p38?-dependent Induction of Interleukin-6
    Article Snippet: A Specific Role for p38 δ in Regulating IL-6 —The family of p38 MAP kinases ( , , , ) consists of four members (α, β, γ, and δ), which differ in their tissue distribution, substrate specificities, and sensitivities to chemical inhibitors such as SB203580 and SB202190, both of which inhibit p38α and p38β ( ).

    Negative Control:

    Article Title: Rabies Virus Stimulates Nitric Oxide Production and CXC Chemokine Ligand 10 Expression in Macrophages through Activation of Extracellular Signal-Regulated Kinases 1 and 2
    Article Snippet: Highly purified bovine serum albumin (BSA; fatty acid free), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), DAPI (4′,6-diamidino-2-phenylindole), 1,4-diazabicyclo-2,2,2-octane, lipopolysaccharide (LPS) from Escherichia coli (serotype O111:B4), bafilomycin A1 (BA1), and ammonium chloride (NH4 Cl) were purchased from Sigma (St. Louis, Mo.). .. PD98059, a selective inhibitor of MAPK/ERK kinase 1 (MEK1); U0126, a potent and specific inhibitor of MEK1/2; U0124, an inactive analogue of U0126 used as a negative control; and SB202190, a specific inhibitor of p38 MAPK, were purchased from EMD Biosciences, Inc. (San Diego, Calif.). .. Rabbit antibodies against phosphorylated forms of ERK1/2 and p38 MAPK were obtained from Santa Cruz Biotechnology (Hercules, Calif.).

    Activity Assay:

    Article Title: Negative regulation of human U6 snRNA promoter by p38 kinase through Oct-1
    Article Snippet: .. Also, in the presence of SB202190 or SB203580, U6 promoter activity was stimulated up to 1.8-fold in transiently transfected cells. .. This stimulated activity was octamer-dependent as deletion or mutation of octamer sequence totally abolished the stimulated activity, strongly suggesting the transcription factor Oct-1 is involved in this stimulation.

    Article Title: The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines
    Article Snippet: Although both analogues significantly decreased activity, a greater effect was observed with D-allose at both 24 and 48 hours compared to 2-DG following treatment (black bars) (Figure A). .. We also investigated the ability of SB202190 to modulate HRE activity (grey bars). ..

    Transfection:

    Article Title: Negative regulation of human U6 snRNA promoter by p38 kinase through Oct-1
    Article Snippet: .. Also, in the presence of SB202190 or SB203580, U6 promoter activity was stimulated up to 1.8-fold in transiently transfected cells. .. This stimulated activity was octamer-dependent as deletion or mutation of octamer sequence totally abolished the stimulated activity, strongly suggesting the transcription factor Oct-1 is involved in this stimulation.

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    • sb202190  (Millipore)
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    Millipore sb202190
    Cytokine‐induced heat‐shock protein 90 α (hsp90 α ) release by pancreatic beta cells was mediated by c‐Jun N‐terminal kinase (JNK) and not p38 mitogen‐activated protein (MAP) kinase. Human β Lox5 cells were treated with media alone or a JNK inhibitor (SP600125) for 6 hr before 24 hr stimulation with interleukin‐1 β (IL‐1 β ), tumour necrosis factor ‐α (TNF‐ α ) and interferon ‐γ (IFN‐ γ ) (Cyt). Extracellular IL‐6 (a) and hsp90 α (b) levels were measured by ELISA. Extracellular hsp90 α levels were calculated relative to hsp90 α release by control cells cultured without SP600125 or cytokines. Extracellular hsp90 α values ranged between 6·8 and 77·1 ng/ml. Human β Lox5 cells were transfected for 72 hr with control or JNK siRNA. JNK knockdown was assessed by immunoblotting (c, d). Transfected cells were then treated with media alone or IL‐1 β , TNF‐ α , and IFN‐ γ (Cyt) for 24 hr. Extracellular hsp90 α levels were analysed by ELISA (e). Data are presented as relative to control siRNA‐transfected cells treated with media alone. Hsp90 α values ranged between 13·1 and 178·5 ng/ml. Human β Lox5 cells were treated with media alone or the p38 MAP kinase inhibitor <t>(SB202190)</t> for 6 hr before 24 hr stimulation with IL‐1 β , TNF‐ α , and IFN‐ γ (Cyt). Extracellular IL‐6 (f) and hsp90 α (g) levels were measured by ELISA. Extracellular hsp90 α levels were indicated relative to control cells treated without SB202190 or cytokines. Extracellular hsp90 α values ranged between 2·8 and 34·1 ng/ml. Data are mean + SEM of n = 3 or n = 4 experiments. * P
    Sb202190, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p38 kinases  (Millipore)
    97
    Millipore p38 kinases
    Effect of ATP and anisomycin on JNK and <t>p38</t> phosphorylation ( a ) and NKCC ( b ) in C11 cells. 100 μM ATP and 0.1 μM anisomycin were added during the last 40 min of preincubation of cells in Cl − -depleted medium. Phosphoprotein content and NKCC activity in the absence of ATP and anisomycin were taken as 1.0 and 100%, respectively. Mean values from three ( a ) or four ( b ) independent experiments are shown
    P38 Kinases, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cytokine‐induced heat‐shock protein 90 α (hsp90 α ) release by pancreatic beta cells was mediated by c‐Jun N‐terminal kinase (JNK) and not p38 mitogen‐activated protein (MAP) kinase. Human β Lox5 cells were treated with media alone or a JNK inhibitor (SP600125) for 6 hr before 24 hr stimulation with interleukin‐1 β (IL‐1 β ), tumour necrosis factor ‐α (TNF‐ α ) and interferon ‐γ (IFN‐ γ ) (Cyt). Extracellular IL‐6 (a) and hsp90 α (b) levels were measured by ELISA. Extracellular hsp90 α levels were calculated relative to hsp90 α release by control cells cultured without SP600125 or cytokines. Extracellular hsp90 α values ranged between 6·8 and 77·1 ng/ml. Human β Lox5 cells were transfected for 72 hr with control or JNK siRNA. JNK knockdown was assessed by immunoblotting (c, d). Transfected cells were then treated with media alone or IL‐1 β , TNF‐ α , and IFN‐ γ (Cyt) for 24 hr. Extracellular hsp90 α levels were analysed by ELISA (e). Data are presented as relative to control siRNA‐transfected cells treated with media alone. Hsp90 α values ranged between 13·1 and 178·5 ng/ml. Human β Lox5 cells were treated with media alone or the p38 MAP kinase inhibitor (SB202190) for 6 hr before 24 hr stimulation with IL‐1 β , TNF‐ α , and IFN‐ γ (Cyt). Extracellular IL‐6 (f) and hsp90 α (g) levels were measured by ELISA. Extracellular hsp90 α levels were indicated relative to control cells treated without SB202190 or cytokines. Extracellular hsp90 α values ranged between 2·8 and 34·1 ng/ml. Data are mean + SEM of n = 3 or n = 4 experiments. * P

    Journal: Immunology

    Article Title: Inflammatory stress of pancreatic beta cells drives release of extracellular heat‐shock protein 90α

    doi: 10.1111/imm.12723

    Figure Lengend Snippet: Cytokine‐induced heat‐shock protein 90 α (hsp90 α ) release by pancreatic beta cells was mediated by c‐Jun N‐terminal kinase (JNK) and not p38 mitogen‐activated protein (MAP) kinase. Human β Lox5 cells were treated with media alone or a JNK inhibitor (SP600125) for 6 hr before 24 hr stimulation with interleukin‐1 β (IL‐1 β ), tumour necrosis factor ‐α (TNF‐ α ) and interferon ‐γ (IFN‐ γ ) (Cyt). Extracellular IL‐6 (a) and hsp90 α (b) levels were measured by ELISA. Extracellular hsp90 α levels were calculated relative to hsp90 α release by control cells cultured without SP600125 or cytokines. Extracellular hsp90 α values ranged between 6·8 and 77·1 ng/ml. Human β Lox5 cells were transfected for 72 hr with control or JNK siRNA. JNK knockdown was assessed by immunoblotting (c, d). Transfected cells were then treated with media alone or IL‐1 β , TNF‐ α , and IFN‐ γ (Cyt) for 24 hr. Extracellular hsp90 α levels were analysed by ELISA (e). Data are presented as relative to control siRNA‐transfected cells treated with media alone. Hsp90 α values ranged between 13·1 and 178·5 ng/ml. Human β Lox5 cells were treated with media alone or the p38 MAP kinase inhibitor (SB202190) for 6 hr before 24 hr stimulation with IL‐1 β , TNF‐ α , and IFN‐ γ (Cyt). Extracellular IL‐6 (f) and hsp90 α (g) levels were measured by ELISA. Extracellular hsp90 α levels were indicated relative to control cells treated without SB202190 or cytokines. Extracellular hsp90 α values ranged between 2·8 and 34·1 ng/ml. Data are mean + SEM of n = 3 or n = 4 experiments. * P

    Article Snippet: Cells were treated with 100 μ m 1400W (Cayman) to inhibit iNOS activity, 10 n m chetomin (Cayman) to inhibit HIF‐1 α activity, 100 μ m dimethyloxaloylglycine (DMOG) (Sigma‐Aldrich) to stabilize HIF‐1 α , 0·2 and 1 m m TUDCA (Millipore, Billierica, MA) to mitigate ER stress, 10 μ m SP600125 (Santa Cruz Biotechnology, Inc., Dallas, TX) to inhibit JNK signalling, or 5 and 10 μ m SB202190 (Sigma‐Aldrich) to inhibit p38 MAPK activation.

    Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection

    Effect of ATP and anisomycin on JNK and p38 phosphorylation ( a ) and NKCC ( b ) in C11 cells. 100 μM ATP and 0.1 μM anisomycin were added during the last 40 min of preincubation of cells in Cl − -depleted medium. Phosphoprotein content and NKCC activity in the absence of ATP and anisomycin were taken as 1.0 and 100%, respectively. Mean values from three ( a ) or four ( b ) independent experiments are shown

    Journal: Purinergic Signalling

    Article Title: Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

    doi: 10.1007/s11302-007-9057-z

    Figure Lengend Snippet: Effect of ATP and anisomycin on JNK and p38 phosphorylation ( a ) and NKCC ( b ) in C11 cells. 100 μM ATP and 0.1 μM anisomycin were added during the last 40 min of preincubation of cells in Cl − -depleted medium. Phosphoprotein content and NKCC activity in the absence of ATP and anisomycin were taken as 1.0 and 100%, respectively. Mean values from three ( a ) or four ( b ) independent experiments are shown

    Article Snippet: Chemicals ATP, ouabain, bumetanide, forskolin, and PMA were obtained from Sigma (St. Louis, MO, USA); anisomycin, cell-permeable inhibitors of JNK and p38 kinases (compounds SP600125 and SB202190, respectively) and their negative controls [N1 -methyl-1,9-pyrazoloanthrone (MPA) and compound SB202474, respectively] were purchased from Calbiochem (La Jolla, CA, USA), and 86 RbCl from Dupont (Boston, MA, USA).

    Techniques: Activity Assay

    Representative blots revealing the effects of SP600125 ( a ), MPA ( b ), SB202190 ( c ), and SB202474 ( d ) on JNK and p38 phosphorylation in C11 cells under control conditions and in the presence of 100 μM ATP or 0.1 μM anisomycin. ATP and anisomycin were added during the last 40 min of preincubation of cells in Cl − -depleted medium. Compounds SP600125, MPA, SB202190, and SB202474 were added at the indicated concentrations 30 min before ATP or anisomycin

    Journal: Purinergic Signalling

    Article Title: Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

    doi: 10.1007/s11302-007-9057-z

    Figure Lengend Snippet: Representative blots revealing the effects of SP600125 ( a ), MPA ( b ), SB202190 ( c ), and SB202474 ( d ) on JNK and p38 phosphorylation in C11 cells under control conditions and in the presence of 100 μM ATP or 0.1 μM anisomycin. ATP and anisomycin were added during the last 40 min of preincubation of cells in Cl − -depleted medium. Compounds SP600125, MPA, SB202190, and SB202474 were added at the indicated concentrations 30 min before ATP or anisomycin

    Article Snippet: Chemicals ATP, ouabain, bumetanide, forskolin, and PMA were obtained from Sigma (St. Louis, MO, USA); anisomycin, cell-permeable inhibitors of JNK and p38 kinases (compounds SP600125 and SB202190, respectively) and their negative controls [N1 -methyl-1,9-pyrazoloanthrone (MPA) and compound SB202474, respectively] were purchased from Calbiochem (La Jolla, CA, USA), and 86 RbCl from Dupont (Boston, MA, USA).

    Techniques: