phospho ikkα β  (Cell Signaling Technology Inc)

 
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  • 91
    Name:
    Phospho IKKα β Ser176 180 Antibody II
    Description:
    The NF κB Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins 1 3 Most agents that activate NF κB do so through a common pathway based on phosphorylation induced proteasome mediated degradation of IκB 3 7 The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase IKK complex whose catalysis is generally carried out by three tightly associated IKK subunits IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit 8 9 Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ Ser176 and Ser180 in IKKα which causes conformational changes resulting in kinase activation 10 13
    Catalog Number:
    2694
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a phosphopeptide corresponding to a region surrounding Ser177/181 of IKKβ. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey
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    Structured Review

    Cell Signaling Technology Inc phospho ikkα β
    Effect of DHMDT on the LPS-induced phosphorylation of <t>IKKα/β,</t> Akt and MAPKs in RAW 264.7 macrophages. The RAW 264.7 cells were pretreated with 800 µg/ml DHMDT for 1 h prior to exposure to LPS for 30 min, and total proteins were isolated. (A) The proteins were subjected to SDS-PAGE, followed by western blot analysis. (B) ImageJ densitometric analysis of bands expressed in relation to β-actin. Data are presented as mean ± standard deviation of the mean. *P
    The NF κB Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins 1 3 Most agents that activate NF κB do so through a common pathway based on phosphorylation induced proteasome mediated degradation of IκB 3 7 The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase IKK complex whose catalysis is generally carried out by three tightly associated IKK subunits IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit 8 9 Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ Ser176 and Ser180 in IKKα which causes conformational changes resulting in kinase activation 10 13
    https://www.bioz.com/result/phospho ikkα β/product/Cell Signaling Technology Inc
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    phospho ikkα β - by Bioz Stars, 2020-09
    91/100 stars

    Images

    1) Product Images from "Anti-inflammatory effects of Daehwangmokdantang, a traditional herbal formulation, in lipopolysaccharide-stimulated RAW 264.7 macrophages"

    Article Title: Anti-inflammatory effects of Daehwangmokdantang, a traditional herbal formulation, in lipopolysaccharide-stimulated RAW 264.7 macrophages

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.5296

    Effect of DHMDT on the LPS-induced phosphorylation of IKKα/β, Akt and MAPKs in RAW 264.7 macrophages. The RAW 264.7 cells were pretreated with 800 µg/ml DHMDT for 1 h prior to exposure to LPS for 30 min, and total proteins were isolated. (A) The proteins were subjected to SDS-PAGE, followed by western blot analysis. (B) ImageJ densitometric analysis of bands expressed in relation to β-actin. Data are presented as mean ± standard deviation of the mean. *P
    Figure Legend Snippet: Effect of DHMDT on the LPS-induced phosphorylation of IKKα/β, Akt and MAPKs in RAW 264.7 macrophages. The RAW 264.7 cells were pretreated with 800 µg/ml DHMDT for 1 h prior to exposure to LPS for 30 min, and total proteins were isolated. (A) The proteins were subjected to SDS-PAGE, followed by western blot analysis. (B) ImageJ densitometric analysis of bands expressed in relation to β-actin. Data are presented as mean ± standard deviation of the mean. *P

    Techniques Used: Isolation, SDS Page, Western Blot, Standard Deviation

    2) Product Images from "Interleukin (IL) 1? Induction of IL-6 Is Mediated by a Novel Phosphatidylinositol 3-Kinase-dependent AKT/I?B Kinase ? Pathway Targeting Activator Protein-1 *"

    Article Title: Interleukin (IL) 1? Induction of IL-6 Is Mediated by a Novel Phosphatidylinositol 3-Kinase-dependent AKT/I?B Kinase ? Pathway Targeting Activator Protein-1 *

    Journal:

    doi: 10.1074/jbc.M707692200

    AP-1 is the target transcription factor of PI 3-kinase/AKT/IKKα. In A , Caco-2 cells were transfected with the IL-6 promoter-specific AP-1 site luciferase reporter (0.25 μg) together with β-galactosidase (0.25 μg). Eighteen
    Figure Legend Snippet: AP-1 is the target transcription factor of PI 3-kinase/AKT/IKKα. In A , Caco-2 cells were transfected with the IL-6 promoter-specific AP-1 site luciferase reporter (0.25 μg) together with β-galactosidase (0.25 μg). Eighteen

    Techniques Used: Transfection, Luciferase

    Interleukin 1 induction of IL-6 promoter activation is dependent on AKT and on the Thr 23 AKT phosphorylation site on IKKα. A , parental IL-6 promoter construct, pIL-6 -luc651 containing 651 bases from the transcription start site. Relevant transcription
    Figure Legend Snippet: Interleukin 1 induction of IL-6 promoter activation is dependent on AKT and on the Thr 23 AKT phosphorylation site on IKKα. A , parental IL-6 promoter construct, pIL-6 -luc651 containing 651 bases from the transcription start site. Relevant transcription

    Techniques Used: Activation Assay, Construct

    3) Product Images from "HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2"

    Article Title: HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022982

    Nef induces NF-κB signalling in an Acidic Cluster dependent manner. Seven days old human primary MDMs were treated for 30′ with 100 ng/ml of myr + wild type (WT) recNef, the protein lacking the first 44 aminoacids (ΔN-Term), mutated in the myristoylation site (G2A) or myr + recNef 4EA mutant. Total cell protein extracts (50 µg) were analyzed by Western Blot as reported in Materials and Methods section using specific anti phospho-IKKα/IKKβ and anti IKKα/IKKβ ( A ) or with anti IκB-α ( B ) antibodies. In both ( A ) and ( B ) β-tubulin steady-steate expression level was used as an internal loading control. Results shown represent one out three different donors.
    Figure Legend Snippet: Nef induces NF-κB signalling in an Acidic Cluster dependent manner. Seven days old human primary MDMs were treated for 30′ with 100 ng/ml of myr + wild type (WT) recNef, the protein lacking the first 44 aminoacids (ΔN-Term), mutated in the myristoylation site (G2A) or myr + recNef 4EA mutant. Total cell protein extracts (50 µg) were analyzed by Western Blot as reported in Materials and Methods section using specific anti phospho-IKKα/IKKβ and anti IKKα/IKKβ ( A ) or with anti IκB-α ( B ) antibodies. In both ( A ) and ( B ) β-tubulin steady-steate expression level was used as an internal loading control. Results shown represent one out three different donors.

    Techniques Used: Mutagenesis, Western Blot, Expressing

    4) Product Images from "Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages"

    Article Title: Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages

    Journal: Foods

    doi: 10.3390/foods9030269

    Schematic model showing the inhibitory effects of compound 1 on lipopolysaccharide-induced inflammatory response in RAW 264.7 mouse macrophages through the inhibition of IKKα/β, I-κBα, NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the activities of IL-6 and TNF-α. 1,5-dimethyl citrate ( 1 ).
    Figure Legend Snippet: Schematic model showing the inhibitory effects of compound 1 on lipopolysaccharide-induced inflammatory response in RAW 264.7 mouse macrophages through the inhibition of IKKα/β, I-κBα, NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the activities of IL-6 and TNF-α. 1,5-dimethyl citrate ( 1 ).

    Techniques Used: Inhibition

    Effects of compound 1 on the LPS-induced expression of the IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), and NF-κB p65 proteins in RAW 264.7 mouse macrophages. ( A ) Representative Western blots of IKKα/β, I-κBα, NF-κB p65, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression. Quantitative graph of ( B ) p-IKKα/β, ( C ) p-I-κBα, and ( D ) p-p65 (mean ± SD, * p
    Figure Legend Snippet: Effects of compound 1 on the LPS-induced expression of the IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), and NF-κB p65 proteins in RAW 264.7 mouse macrophages. ( A ) Representative Western blots of IKKα/β, I-κBα, NF-κB p65, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression. Quantitative graph of ( B ) p-IKKα/β, ( C ) p-I-κBα, and ( D ) p-p65 (mean ± SD, * p

    Techniques Used: Expressing, Western Blot

    5) Product Images from "Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-?B in Human Brain Endothelial Cells"

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-?B in Human Brain Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035862

    Effects of CAPE on IbeA+ E. coli K1-induced NF-κB activation and pathogenicities in vitro and in vivo . ( A ) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the Materials and Methods . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in Methods and Materials . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P
    Figure Legend Snippet: Effects of CAPE on IbeA+ E. coli K1-induced NF-κB activation and pathogenicities in vitro and in vivo . ( A ) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the Materials and Methods . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in Methods and Materials . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P

    Techniques Used: Activation Assay, In Vitro, In Vivo, Incubation, Transmigration Assay, Positive Control, Mouse Assay

    Role of vimentin in IbeA+ E. coli K1-induced NF-κB activation. ( A ) Immunofluorescence microscopy was used to examine the correlation between vimentin reorganization and NF-κB translocation to the nucleus after 2 h of stimulation with IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization of vimentin and NF-κB (p65) Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced NF-κB activation in HBMECs by siRNA-mediated knockdown of vimentin. HBMECs were transfected with vimentin or control siRNA as described in Materials and Methods . After 24 h incubation, the cells were treated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. Vimentin (VIM), α7 nAChR, ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, and PSF re-localization were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; VIM KD: HBMECs transfected with vimentin siRNA; UNT: Untreated HBMECs.
    Figure Legend Snippet: Role of vimentin in IbeA+ E. coli K1-induced NF-κB activation. ( A ) Immunofluorescence microscopy was used to examine the correlation between vimentin reorganization and NF-κB translocation to the nucleus after 2 h of stimulation with IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization of vimentin and NF-κB (p65) Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced NF-κB activation in HBMECs by siRNA-mediated knockdown of vimentin. HBMECs were transfected with vimentin or control siRNA as described in Materials and Methods . After 24 h incubation, the cells were treated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. Vimentin (VIM), α7 nAChR, ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, and PSF re-localization were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; VIM KD: HBMECs transfected with vimentin siRNA; UNT: Untreated HBMECs.

    Techniques Used: Activation Assay, Immunofluorescence, Microscopy, Translocation Assay, Staining, Transfection, Incubation

    Effects of vimentin head domain deletion on IbeA-induced NF-κB activation and interaction with β-tubulin. ( A ) The cytoplasmic fractions of the GFP–VRT, GFP-VH and GFP transductants were extracted and immunoprecipitated (IP) using the mouse anti-GFP antibody as described in Materials and Methods . The GFP-IP complexes were subjected to Western blotting using the rabbit polyclonal antibodies against GFP, NF-κB (P65), and β-tubulin. Band a, GFP–VRT (72 kDa); band b, GFP-VH (37 kDa); band c, GFP (27 kDa); band d, NF-κB (P65), (65 kDa); and band e, β-tubulin, (50 kDa). ( B ) Immunofluorescence images of the GFP–VRT and GFP transductants incubated with or without the IbeA protein (0.1 µg/ml) for 2 h. The cells were double-stained with the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). Arrows indicate cells with NF-κB (P65) translocation to the nucleus, which was increased in the GFP transductants and reduced in GFP-VRT-transduced HBMECs upon stimulation with IbeA. Scale bar, 50 µm. ( C ) Western blot of the transduced HBMECs treated with the IbeA protein (0.1 µg/ml). ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, vimentin (VIM), GFP and PSF re-localization were examined in cytoplasmic fractions after 30 min of IbeA stimulation. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of IbeA incubation. β-actin in both fractions was detected as internal loading controls.
    Figure Legend Snippet: Effects of vimentin head domain deletion on IbeA-induced NF-κB activation and interaction with β-tubulin. ( A ) The cytoplasmic fractions of the GFP–VRT, GFP-VH and GFP transductants were extracted and immunoprecipitated (IP) using the mouse anti-GFP antibody as described in Materials and Methods . The GFP-IP complexes were subjected to Western blotting using the rabbit polyclonal antibodies against GFP, NF-κB (P65), and β-tubulin. Band a, GFP–VRT (72 kDa); band b, GFP-VH (37 kDa); band c, GFP (27 kDa); band d, NF-κB (P65), (65 kDa); and band e, β-tubulin, (50 kDa). ( B ) Immunofluorescence images of the GFP–VRT and GFP transductants incubated with or without the IbeA protein (0.1 µg/ml) for 2 h. The cells were double-stained with the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). Arrows indicate cells with NF-κB (P65) translocation to the nucleus, which was increased in the GFP transductants and reduced in GFP-VRT-transduced HBMECs upon stimulation with IbeA. Scale bar, 50 µm. ( C ) Western blot of the transduced HBMECs treated with the IbeA protein (0.1 µg/ml). ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, vimentin (VIM), GFP and PSF re-localization were examined in cytoplasmic fractions after 30 min of IbeA stimulation. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of IbeA incubation. β-actin in both fractions was detected as internal loading controls.

    Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Incubation, Staining, Translocation Assay

    β-tublulin is required for IbeA+ E. coli K1-induced NF-κB activation. ( A ) IbeA− and IbeA+ E. coli K1-induced β-tubulin/vimentin clustering and colocalization. Immunofluorescence microscopy was used to examine the clustering and reorganization of vimentin and β-tubulin after 2 h of incubation with the IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against β-tubulin conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization between vimentin and β-tubulin around the perinuclear region. Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced cytoplasmic activation and nuclear translocation of NF-κB (p65) in HBMECs by the microtubule inhibitors. HBMECs were incubated with or without colchicines (Col, 2 µM), vincristine (Vin, 1 µM), nocodazole (Noc, 25 µg/ml) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). Phosphorylation of ERK1/2 (p-Erk1/2) and IKK α/β (p-IKK α/β) was examined in cytoplasmic fractions after 30 min of E. coli K1 treatment. NF-κB (p65) translocation to nucleus in nuclear fractions was examined after 2 h of E. coli K1 incubation. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.
    Figure Legend Snippet: β-tublulin is required for IbeA+ E. coli K1-induced NF-κB activation. ( A ) IbeA− and IbeA+ E. coli K1-induced β-tubulin/vimentin clustering and colocalization. Immunofluorescence microscopy was used to examine the clustering and reorganization of vimentin and β-tubulin after 2 h of incubation with the IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against β-tubulin conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization between vimentin and β-tubulin around the perinuclear region. Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced cytoplasmic activation and nuclear translocation of NF-κB (p65) in HBMECs by the microtubule inhibitors. HBMECs were incubated with or without colchicines (Col, 2 µM), vincristine (Vin, 1 µM), nocodazole (Noc, 25 µg/ml) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). Phosphorylation of ERK1/2 (p-Erk1/2) and IKK α/β (p-IKK α/β) was examined in cytoplasmic fractions after 30 min of E. coli K1 treatment. NF-κB (p65) translocation to nucleus in nuclear fractions was examined after 2 h of E. coli K1 incubation. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

    Techniques Used: Activation Assay, Immunofluorescence, Microscopy, Incubation, Staining, Translocation Assay

    Inhibition of IbeA+ E. coli -induced IKK phosphorylation and NF-κB activation by MEK/ERK inhibitors. ( A ) HBMECs were incubated with or without PD098059 (50 µM) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). ( B ) HBMECs were incubated with or without ERK89 (vimentin-binding domain, 25 µg/ml) and ERK312 (control peptide, 25 µg/ml) for 60 min before infection with E44 or ZD1 (10 7 /ml). In both ( A ) and ( B ), ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β) and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of infection with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.
    Figure Legend Snippet: Inhibition of IbeA+ E. coli -induced IKK phosphorylation and NF-κB activation by MEK/ERK inhibitors. ( A ) HBMECs were incubated with or without PD098059 (50 µM) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). ( B ) HBMECs were incubated with or without ERK89 (vimentin-binding domain, 25 µg/ml) and ERK312 (control peptide, 25 µg/ml) for 60 min before infection with E44 or ZD1 (10 7 /ml). In both ( A ) and ( B ), ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β) and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of infection with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

    Techniques Used: Inhibition, Activation Assay, Incubation, Binding Assay, Infection, Translocation Assay

    6) Product Images from "Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation"

    Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06407-11

    TAK1 interaction with TAB1 controls CARMA1-dependent ubiquitination and degradation of BCL10. (A to D) 293T cells were cotransfected for 48 h with combinations of expression vectors for the proteins indicated at the top of each blot. WCLs were prepared
    Figure Legend Snippet: TAK1 interaction with TAB1 controls CARMA1-dependent ubiquitination and degradation of BCL10. (A to D) 293T cells were cotransfected for 48 h with combinations of expression vectors for the proteins indicated at the top of each blot. WCLs were prepared

    Techniques Used: Expressing

    7) Product Images from "Innate Sensing of HIV-1 Assembly by Tetherin Induces NF?B-Dependent Proinflammatory Responses"

    Article Title: Innate Sensing of HIV-1 Assembly by Tetherin Induces NF?B-Dependent Proinflammatory Responses

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2012.10.007

    Tetherin Induces NFκB-Dependent Responses upon Overexpression, Crosslinking, and Restriction of Virion Release (A) Fold activation of a firefly-luciferase NFκB reporter gene in 293 cells transiently cotransfected with tetherin, MAVS, or control YFP vectors. (B) Fold activation of the same reporter in 293 or 293THN cells treated for 24 hr with a rabbit anti-tetherin polyclonal serum and a secondary anti-rabbit antibody. (C) Time course of endogenous IκB degradation and IKKα/β phosphorylation in 293THN cells after antibody crosslinking. (D and E) (D) Fold increases in NFκB-reporter activity in 293 and 293THN cells transfected with wild-type and Vpu(−) HIV-1 proviruses and (E) fold changes in Cxcl10 mRNA levels compared to YFP transfection calculated relative to Gapdh by qRT-PCR. (F and G) NFκB-reporter fold activation in 293 and 293THN cells transfected with GFP-fused Ebolavirus VP40 expression vector (F) or MLV provirus or derivatives (MLVΔPY and MLVΔPY/p6) (G). Fold changes relative to 293 cells transfected with YFP control (A, D–G) or 293 nontreated cells (B). ∗ p > 0.05 and ∗∗∗ p > 0.001 as determined by two-tailed t test. All error bars represent ±SEM of three independent experiments.
    Figure Legend Snippet: Tetherin Induces NFκB-Dependent Responses upon Overexpression, Crosslinking, and Restriction of Virion Release (A) Fold activation of a firefly-luciferase NFκB reporter gene in 293 cells transiently cotransfected with tetherin, MAVS, or control YFP vectors. (B) Fold activation of the same reporter in 293 or 293THN cells treated for 24 hr with a rabbit anti-tetherin polyclonal serum and a secondary anti-rabbit antibody. (C) Time course of endogenous IκB degradation and IKKα/β phosphorylation in 293THN cells after antibody crosslinking. (D and E) (D) Fold increases in NFκB-reporter activity in 293 and 293THN cells transfected with wild-type and Vpu(−) HIV-1 proviruses and (E) fold changes in Cxcl10 mRNA levels compared to YFP transfection calculated relative to Gapdh by qRT-PCR. (F and G) NFκB-reporter fold activation in 293 and 293THN cells transfected with GFP-fused Ebolavirus VP40 expression vector (F) or MLV provirus or derivatives (MLVΔPY and MLVΔPY/p6) (G). Fold changes relative to 293 cells transfected with YFP control (A, D–G) or 293 nontreated cells (B). ∗ p > 0.05 and ∗∗∗ p > 0.001 as determined by two-tailed t test. All error bars represent ±SEM of three independent experiments.

    Techniques Used: Over Expression, Activation Assay, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Expressing, Plasmid Preparation, Two Tailed Test

    8) Product Images from "PEROXYNITRITE IS A POTENT INHIBITOR OF NF-? B ACTIVATION TRIGGERED BY INFLAMMATORY STIMULI IN CARDIAC AND ENDOTHELIAL CELL LINES"

    Article Title: PEROXYNITRITE IS A POTENT INHIBITOR OF NF-? B ACTIVATION TRIGGERED BY INFLAMMATORY STIMULI IN CARDIAC AND ENDOTHELIAL CELL LINES

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M501977200

    A model for peroxynitrite-mediated interference with the NF-κB pathway. Immune stimuli activate the canonical NF-κB pathway, by inducing phosphorylation and activation of IKKβ, in a process regulated by IKKγ. In turn, IKKβ phosphorylates IκBα, leading to its proteasomal degradation. NF-κB dimers (mostly p50/p65) then translocate into the nucleus, bind to κB consensus sequences, and activate the transcription of target genes. In the presence of peroxynitrite, IKKβ phosphorylation is impaired, thereby blocking IKKβ activation and downstream signaling (as indicated by the (−) signs). At the same time, peroxynitrite activates the phosphorylation of IKKα, most probably via the phosphorylation and activation of the upstream kinase NIK. The cellular targets of IKKα in this model remain to be defined.
    Figure Legend Snippet: A model for peroxynitrite-mediated interference with the NF-κB pathway. Immune stimuli activate the canonical NF-κB pathway, by inducing phosphorylation and activation of IKKβ, in a process regulated by IKKγ. In turn, IKKβ phosphorylates IκBα, leading to its proteasomal degradation. NF-κB dimers (mostly p50/p65) then translocate into the nucleus, bind to κB consensus sequences, and activate the transcription of target genes. In the presence of peroxynitrite, IKKβ phosphorylation is impaired, thereby blocking IKKβ activation and downstream signaling (as indicated by the (−) signs). At the same time, peroxynitrite activates the phosphorylation of IKKα, most probably via the phosphorylation and activation of the upstream kinase NIK. The cellular targets of IKKα in this model remain to be defined.

    Techniques Used: Activation Assay, Blocking Assay

    Peroxynitrite inhibits IKK kinase activity by preventing specifically the phosphorylation of IKKβ in H9C2 cells. A and B: IKKkinase assay (KA) in cells treated with LPS or TNFα. LPS strongly activated IKK kinase from 10 to 30 minutes (A), whereas the effects of TNFα were most pronounced at 5 and 10 minutes (B). The activation of IKK was associated with an increased phosphorylation of IKKα and IKKβ by LPS and TNFα. IKKα levels are shown as an internal control. C and D: IKK kinase activity in cells pretreated with peroxynitrite (PN, 250 μM, 20 min) and stimulated with LPS (C, 20 min) or TNFα (D, 10 min). PN abrogated IKK activity triggered by LPS or TNFα. E and F: Phosphorylation of IKKα and β was induced both by LPS (20 min, E) and TNFα (10 min, F). PN completely prevented the phosphorylation of IKKβ triggered by LPS and TNFα, whereas it did not reduce IKKα phosphorylation in response to LPS and TNFα. No significant changes in the levels of IKKα, IKKβ and α-tubulin were found between the different conditions. G and H: densitometric analysis of phospho-IKK western blots showing mean±sem of 3 observations. NS: Not Stimulated. * p
    Figure Legend Snippet: Peroxynitrite inhibits IKK kinase activity by preventing specifically the phosphorylation of IKKβ in H9C2 cells. A and B: IKKkinase assay (KA) in cells treated with LPS or TNFα. LPS strongly activated IKK kinase from 10 to 30 minutes (A), whereas the effects of TNFα were most pronounced at 5 and 10 minutes (B). The activation of IKK was associated with an increased phosphorylation of IKKα and IKKβ by LPS and TNFα. IKKα levels are shown as an internal control. C and D: IKK kinase activity in cells pretreated with peroxynitrite (PN, 250 μM, 20 min) and stimulated with LPS (C, 20 min) or TNFα (D, 10 min). PN abrogated IKK activity triggered by LPS or TNFα. E and F: Phosphorylation of IKKα and β was induced both by LPS (20 min, E) and TNFα (10 min, F). PN completely prevented the phosphorylation of IKKβ triggered by LPS and TNFα, whereas it did not reduce IKKα phosphorylation in response to LPS and TNFα. No significant changes in the levels of IKKα, IKKβ and α-tubulin were found between the different conditions. G and H: densitometric analysis of phospho-IKK western blots showing mean±sem of 3 observations. NS: Not Stimulated. * p

    Techniques Used: Activity Assay, Activation Assay, Western Blot

    9) Product Images from "PEROXYNITRITE IS A POTENT INHIBITOR OF NF-? B ACTIVATION TRIGGERED BY INFLAMMATORY STIMULI IN CARDIAC AND ENDOTHELIAL CELL LINES"

    Article Title: PEROXYNITRITE IS A POTENT INHIBITOR OF NF-? B ACTIVATION TRIGGERED BY INFLAMMATORY STIMULI IN CARDIAC AND ENDOTHELIAL CELL LINES

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M501977200

    A model for peroxynitrite-mediated interference with the NF-κB pathway. Immune stimuli activate the canonical NF-κB pathway, by inducing phosphorylation and activation of IKKβ, in a process regulated by IKKγ. In turn, IKKβ phosphorylates IκBα, leading to its proteasomal degradation. NF-κB dimers (mostly p50/p65) then translocate into the nucleus, bind to κB consensus sequences, and activate the transcription of target genes. In the presence of peroxynitrite, IKKβ phosphorylation is impaired, thereby blocking IKKβ activation and downstream signaling (as indicated by the (−) signs). At the same time, peroxynitrite activates the phosphorylation of IKKα, most probably via the phosphorylation and activation of the upstream kinase NIK. The cellular targets of IKKα in this model remain to be defined.
    Figure Legend Snippet: A model for peroxynitrite-mediated interference with the NF-κB pathway. Immune stimuli activate the canonical NF-κB pathway, by inducing phosphorylation and activation of IKKβ, in a process regulated by IKKγ. In turn, IKKβ phosphorylates IκBα, leading to its proteasomal degradation. NF-κB dimers (mostly p50/p65) then translocate into the nucleus, bind to κB consensus sequences, and activate the transcription of target genes. In the presence of peroxynitrite, IKKβ phosphorylation is impaired, thereby blocking IKKβ activation and downstream signaling (as indicated by the (−) signs). At the same time, peroxynitrite activates the phosphorylation of IKKα, most probably via the phosphorylation and activation of the upstream kinase NIK. The cellular targets of IKKα in this model remain to be defined.

    Techniques Used: Activation Assay, Blocking Assay

    PN activates the phosphorylation of IKKα and NIK. IKKα phosphorylation was monitored in cells stimulated with LPS (A) or TNFα (B) for 10 to 120 min, in the absence or presence of peroxynitrite (PN). In the presence of PN, there was a strong phosphorylation of IKKα, maintained for up to 120 min after LPS or TNFα, with no change in total IKKα. C: PN alone strongly activated the phosphorylation of IKKα and NIK, an effect starting after 10 min. NS: Not Stimulated. NSB: non specific band
    Figure Legend Snippet: PN activates the phosphorylation of IKKα and NIK. IKKα phosphorylation was monitored in cells stimulated with LPS (A) or TNFα (B) for 10 to 120 min, in the absence or presence of peroxynitrite (PN). In the presence of PN, there was a strong phosphorylation of IKKα, maintained for up to 120 min after LPS or TNFα, with no change in total IKKα. C: PN alone strongly activated the phosphorylation of IKKα and NIK, an effect starting after 10 min. NS: Not Stimulated. NSB: non specific band

    Techniques Used:

    Peroxynitrite inhibits IKK kinase activity by preventing specifically the phosphorylation of IKKβ in H9C2 cells. A and B: IKKkinase assay (KA) in cells treated with LPS or TNFα. LPS strongly activated IKK kinase from 10 to 30 minutes (A), whereas the effects of TNFα were most pronounced at 5 and 10 minutes (B). The activation of IKK was associated with an increased phosphorylation of IKKα and IKKβ by LPS and TNFα. IKKα levels are shown as an internal control. C and D: IKK kinase activity in cells pretreated with peroxynitrite (PN, 250 μM, 20 min) and stimulated with LPS (C, 20 min) or TNFα (D, 10 min). PN abrogated IKK activity triggered by LPS or TNFα. E and F: Phosphorylation of IKKα and β was induced both by LPS (20 min, E) and TNFα (10 min, F). PN completely prevented the phosphorylation of IKKβ triggered by LPS and TNFα, whereas it did not reduce IKKα phosphorylation in response to LPS and TNFα. No significant changes in the levels of IKKα, IKKβ and α-tubulin were found between the different conditions. G and H: densitometric analysis of phospho-IKK western blots showing mean±sem of 3 observations. NS: Not Stimulated. * p
    Figure Legend Snippet: Peroxynitrite inhibits IKK kinase activity by preventing specifically the phosphorylation of IKKβ in H9C2 cells. A and B: IKKkinase assay (KA) in cells treated with LPS or TNFα. LPS strongly activated IKK kinase from 10 to 30 minutes (A), whereas the effects of TNFα were most pronounced at 5 and 10 minutes (B). The activation of IKK was associated with an increased phosphorylation of IKKα and IKKβ by LPS and TNFα. IKKα levels are shown as an internal control. C and D: IKK kinase activity in cells pretreated with peroxynitrite (PN, 250 μM, 20 min) and stimulated with LPS (C, 20 min) or TNFα (D, 10 min). PN abrogated IKK activity triggered by LPS or TNFα. E and F: Phosphorylation of IKKα and β was induced both by LPS (20 min, E) and TNFα (10 min, F). PN completely prevented the phosphorylation of IKKβ triggered by LPS and TNFα, whereas it did not reduce IKKα phosphorylation in response to LPS and TNFα. No significant changes in the levels of IKKα, IKKβ and α-tubulin were found between the different conditions. G and H: densitometric analysis of phospho-IKK western blots showing mean±sem of 3 observations. NS: Not Stimulated. * p

    Techniques Used: Activity Assay, Activation Assay, Western Blot

    10) Product Images from "Experimental chronic kidney disease attenuates ischemia-reperfusion injury in an ex vivo rat lung model"

    Article Title: Experimental chronic kidney disease attenuates ischemia-reperfusion injury in an ex vivo rat lung model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171736

    Effects of chronic kidney disease on NF-κB activation. (A) Cytoplasmic levels of phosphorylated IKK; (B) nuclear levels of total NF-κB p65; and (C) IκB-α level. The data are expressed as the means ± SD. *Significantly different from the control ( P
    Figure Legend Snippet: Effects of chronic kidney disease on NF-κB activation. (A) Cytoplasmic levels of phosphorylated IKK; (B) nuclear levels of total NF-κB p65; and (C) IκB-α level. The data are expressed as the means ± SD. *Significantly different from the control ( P

    Techniques Used: Activation Assay

    11) Product Images from "HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2"

    Article Title: HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022982

    Nef induces NF-κB signalling in an Acidic Cluster dependent manner. Seven days old human primary MDMs were treated for 30′ with 100 ng/ml of myr + wild type (WT) recNef, the protein lacking the first 44 aminoacids (ΔN-Term), mutated in the myristoylation site (G2A) or myr + recNef 4EA mutant. Total cell protein extracts (50 µg) were analyzed by Western Blot as reported in Materials and Methods section using specific anti phospho-IKKα/IKKβ and anti IKKα/IKKβ ( A ) or with anti IκB-α ( B ) antibodies. In both ( A ) and ( B ) β-tubulin steady-steate expression level was used as an internal loading control. Results shown represent one out three different donors.
    Figure Legend Snippet: Nef induces NF-κB signalling in an Acidic Cluster dependent manner. Seven days old human primary MDMs were treated for 30′ with 100 ng/ml of myr + wild type (WT) recNef, the protein lacking the first 44 aminoacids (ΔN-Term), mutated in the myristoylation site (G2A) or myr + recNef 4EA mutant. Total cell protein extracts (50 µg) were analyzed by Western Blot as reported in Materials and Methods section using specific anti phospho-IKKα/IKKβ and anti IKKα/IKKβ ( A ) or with anti IκB-α ( B ) antibodies. In both ( A ) and ( B ) β-tubulin steady-steate expression level was used as an internal loading control. Results shown represent one out three different donors.

    Techniques Used: Mutagenesis, Western Blot, Expressing

    12) Product Images from "PEROXYNITRITE IS A POTENT INHIBITOR OF NF-? B ACTIVATION TRIGGERED BY INFLAMMATORY STIMULI IN CARDIAC AND ENDOTHELIAL CELL LINES"

    Article Title: PEROXYNITRITE IS A POTENT INHIBITOR OF NF-? B ACTIVATION TRIGGERED BY INFLAMMATORY STIMULI IN CARDIAC AND ENDOTHELIAL CELL LINES

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M501977200

    A model for peroxynitrite-mediated interference with the NF-κB pathway. Immune stimuli activate the canonical NF-κB pathway, by inducing phosphorylation and activation of IKKβ, in a process regulated by IKKγ. In turn, IKKβ phosphorylates IκBα, leading to its proteasomal degradation. NF-κB dimers (mostly p50/p65) then translocate into the nucleus, bind to κB consensus sequences, and activate the transcription of target genes. In the presence of peroxynitrite, IKKβ phosphorylation is impaired, thereby blocking IKKβ activation and downstream signaling (as indicated by the (−) signs). At the same time, peroxynitrite activates the phosphorylation of IKKα, most probably via the phosphorylation and activation of the upstream kinase NIK. The cellular targets of IKKα in this model remain to be defined.
    Figure Legend Snippet: A model for peroxynitrite-mediated interference with the NF-κB pathway. Immune stimuli activate the canonical NF-κB pathway, by inducing phosphorylation and activation of IKKβ, in a process regulated by IKKγ. In turn, IKKβ phosphorylates IκBα, leading to its proteasomal degradation. NF-κB dimers (mostly p50/p65) then translocate into the nucleus, bind to κB consensus sequences, and activate the transcription of target genes. In the presence of peroxynitrite, IKKβ phosphorylation is impaired, thereby blocking IKKβ activation and downstream signaling (as indicated by the (−) signs). At the same time, peroxynitrite activates the phosphorylation of IKKα, most probably via the phosphorylation and activation of the upstream kinase NIK. The cellular targets of IKKα in this model remain to be defined.

    Techniques Used: Activation Assay, Blocking Assay

    PN activates the phosphorylation of IKKα and NIK. IKKα phosphorylation was monitored in cells stimulated with LPS (A) or TNFα (B) for 10 to 120 min, in the absence or presence of peroxynitrite (PN). In the presence of PN, there was a strong phosphorylation of IKKα, maintained for up to 120 min after LPS or TNFα, with no change in total IKKα. C: PN alone strongly activated the phosphorylation of IKKα and NIK, an effect starting after 10 min. NS: Not Stimulated. NSB: non specific band
    Figure Legend Snippet: PN activates the phosphorylation of IKKα and NIK. IKKα phosphorylation was monitored in cells stimulated with LPS (A) or TNFα (B) for 10 to 120 min, in the absence or presence of peroxynitrite (PN). In the presence of PN, there was a strong phosphorylation of IKKα, maintained for up to 120 min after LPS or TNFα, with no change in total IKKα. C: PN alone strongly activated the phosphorylation of IKKα and NIK, an effect starting after 10 min. NS: Not Stimulated. NSB: non specific band

    Techniques Used:

    Peroxynitrite inhibits IKK kinase activity by preventing specifically the phosphorylation of IKKβ in H9C2 cells. A and B: IKKkinase assay (KA) in cells treated with LPS or TNFα. LPS strongly activated IKK kinase from 10 to 30 minutes (A), whereas the effects of TNFα were most pronounced at 5 and 10 minutes (B). The activation of IKK was associated with an increased phosphorylation of IKKα and IKKβ by LPS and TNFα. IKKα levels are shown as an internal control. C and D: IKK kinase activity in cells pretreated with peroxynitrite (PN, 250 μM, 20 min) and stimulated with LPS (C, 20 min) or TNFα (D, 10 min). PN abrogated IKK activity triggered by LPS or TNFα. E and F: Phosphorylation of IKKα and β was induced both by LPS (20 min, E) and TNFα (10 min, F). PN completely prevented the phosphorylation of IKKβ triggered by LPS and TNFα, whereas it did not reduce IKKα phosphorylation in response to LPS and TNFα. No significant changes in the levels of IKKα, IKKβ and α-tubulin were found between the different conditions. G and H: densitometric analysis of phospho-IKK western blots showing mean±sem of 3 observations. NS: Not Stimulated. * p
    Figure Legend Snippet: Peroxynitrite inhibits IKK kinase activity by preventing specifically the phosphorylation of IKKβ in H9C2 cells. A and B: IKKkinase assay (KA) in cells treated with LPS or TNFα. LPS strongly activated IKK kinase from 10 to 30 minutes (A), whereas the effects of TNFα were most pronounced at 5 and 10 minutes (B). The activation of IKK was associated with an increased phosphorylation of IKKα and IKKβ by LPS and TNFα. IKKα levels are shown as an internal control. C and D: IKK kinase activity in cells pretreated with peroxynitrite (PN, 250 μM, 20 min) and stimulated with LPS (C, 20 min) or TNFα (D, 10 min). PN abrogated IKK activity triggered by LPS or TNFα. E and F: Phosphorylation of IKKα and β was induced both by LPS (20 min, E) and TNFα (10 min, F). PN completely prevented the phosphorylation of IKKβ triggered by LPS and TNFα, whereas it did not reduce IKKα phosphorylation in response to LPS and TNFα. No significant changes in the levels of IKKα, IKKβ and α-tubulin were found between the different conditions. G and H: densitometric analysis of phospho-IKK western blots showing mean±sem of 3 observations. NS: Not Stimulated. * p

    Techniques Used: Activity Assay, Activation Assay, Western Blot

    13) Product Images from "Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis"

    Article Title: Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00230

    METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p
    Figure Legend Snippet: METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p

    Techniques Used: Expressing, Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    14) Product Images from "Bacteroides fragilis Enterotoxin Induces Human ?-Defensin-2 Expression in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase/I?B Kinase/NF-?B-Dependent Pathway ▿"

    Article Title: Bacteroides fragilis Enterotoxin Induces Human ?-Defensin-2 Expression in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase/I?B Kinase/NF-?B-Dependent Pathway ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00118-10

    Suppression of IKK activity by siRNA in HT-29 intestinal epithelial cells stimulated with BFT. (A) HT-29 cells were stimulated with BFT (300 ng/ml) for the indicated periods. Phosphorylation and protein expression of IKK-α, IKK-β, and
    Figure Legend Snippet: Suppression of IKK activity by siRNA in HT-29 intestinal epithelial cells stimulated with BFT. (A) HT-29 cells were stimulated with BFT (300 ng/ml) for the indicated periods. Phosphorylation and protein expression of IKK-α, IKK-β, and

    Techniques Used: Activity Assay, Expressing

    15) Product Images from "Loss of glutathione peroxidase 7 promotes TNF-α-induced NF-κB activation in Barrett’s carcinogenesis"

    Article Title: Loss of glutathione peroxidase 7 promotes TNF-α-induced NF-κB activation in Barrett’s carcinogenesis

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgu083

    GPX7 deregulates protein stability of TNFR1 and TRAF2. ( A and B ) Western blot analysis of TNFR1 and TRAF2 protein levels in FLO-1 cells stably expressing GPX7 (GPX7) or control (PcDNA) ( A ) or transiently expressing GPX7 (Ad-GPX7) or control (Ad-CTRL)
    Figure Legend Snippet: GPX7 deregulates protein stability of TNFR1 and TRAF2. ( A and B ) Western blot analysis of TNFR1 and TRAF2 protein levels in FLO-1 cells stably expressing GPX7 (GPX7) or control (PcDNA) ( A ) or transiently expressing GPX7 (Ad-GPX7) or control (Ad-CTRL)

    Techniques Used: Western Blot, Stable Transfection, Expressing

    16) Product Images from "Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis"

    Article Title: Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00230

    METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p
    Figure Legend Snippet: METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p

    Techniques Used: Expressing, Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    17) Product Images from "Tumor necrosis factor-? suppresses angiotensinogen expression through formation of a p50/p50 homodimer in human renal proximal tubular cells"

    Article Title: Tumor necrosis factor-? suppresses angiotensinogen expression through formation of a p50/p50 homodimer in human renal proximal tubular cells

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00078.2010

    DNA binding activities of NF-κB subunits complexes induced by TNF-α treatment. HK-2 cells were treated with 20 ng/ml TNF-α for 0–90 min. After treatment, the binding activities of NF-κB subunits to DNA were determined using EMSA. Bands were identified by supershift assay using anti-p50, anti-p52, and anti-p65 antibodies. *Supershift bands by antibodies. C, control group; T, TNF-α-treated group; N, negative control (without any protein).
    Figure Legend Snippet: DNA binding activities of NF-κB subunits complexes induced by TNF-α treatment. HK-2 cells were treated with 20 ng/ml TNF-α for 0–90 min. After treatment, the binding activities of NF-κB subunits to DNA were determined using EMSA. Bands were identified by supershift assay using anti-p50, anti-p52, and anti-p65 antibodies. *Supershift bands by antibodies. C, control group; T, TNF-α-treated group; N, negative control (without any protein).

    Techniques Used: Binding Assay, Negative Control

    Translocalization of nuclear factor (NF)-κB subunits by TNF-α treatment. Translocalization of NF-κB subunits were examined by Western blot analysis using isolated nuclear protein after treatment with 20 ng/ml TNF-α from 0 to 90 min. Nuclear p50 ( A , n = 3), p52 ( B , n = 3), and p65 ( C , n = 3) levels were normalized based on expression level of lamin A/C. Open columns, control groups; filled columns, TNF-α-treated groups. Data are means ± SD. *Significant difference compared with each time point control ( P
    Figure Legend Snippet: Translocalization of nuclear factor (NF)-κB subunits by TNF-α treatment. Translocalization of NF-κB subunits were examined by Western blot analysis using isolated nuclear protein after treatment with 20 ng/ml TNF-α from 0 to 90 min. Nuclear p50 ( A , n = 3), p52 ( B , n = 3), and p65 ( C , n = 3) levels were normalized based on expression level of lamin A/C. Open columns, control groups; filled columns, TNF-α-treated groups. Data are means ± SD. *Significant difference compared with each time point control ( P

    Techniques Used: Western Blot, Isolation, Expressing

    Schematic summary of AGT suppression by TNF-α in human RPTCs. TNF-α facilitates formation of a p50/p65 heterodimer and a p50/p50 homodimer via a TNFR. After these dimers transfer to nuclei, the excess p50/p50 homodimer inhibits AGT mRNA transcription.
    Figure Legend Snippet: Schematic summary of AGT suppression by TNF-α in human RPTCs. TNF-α facilitates formation of a p50/p65 heterodimer and a p50/p50 homodimer via a TNFR. After these dimers transfer to nuclei, the excess p50/p50 homodimer inhibits AGT mRNA transcription.

    Techniques Used:

    Phosphorylation of IKB kinase (IKK)α/β, IκBα, p105, and p65 by TNF-α treatment. HK-2 cells were treated with 20 ng/ml TNF-α for 0–90 min. After treatment, IKKα/β phosphorylation levels ( A , n = 4), IκBα phosphorylation levels ( B , n = 4), p105 phosphorylation levels ( C , n = 4), and p65 phosphorylation levels ( D , n = 4) were evaluated by Western blot analysis. Phosphorylation levels of IKKα/β and p65 were normalized based on combination of total IKK α with total IKK β levels and total p65, respectively. Phosphorylation levels of IκBα and p105 were normalized based on β-actin levels because of instability of total IκBα and total p105 levels. Open columns, control groups; filled columns, TNF-α-treated groups. Data are means ± SD. *Significant difference compared with each time point control ( P
    Figure Legend Snippet: Phosphorylation of IKB kinase (IKK)α/β, IκBα, p105, and p65 by TNF-α treatment. HK-2 cells were treated with 20 ng/ml TNF-α for 0–90 min. After treatment, IKKα/β phosphorylation levels ( A , n = 4), IκBα phosphorylation levels ( B , n = 4), p105 phosphorylation levels ( C , n = 4), and p65 phosphorylation levels ( D , n = 4) were evaluated by Western blot analysis. Phosphorylation levels of IKKα/β and p65 were normalized based on combination of total IKK α with total IKK β levels and total p65, respectively. Phosphorylation levels of IκBα and p105 were normalized based on β-actin levels because of instability of total IκBα and total p105 levels. Open columns, control groups; filled columns, TNF-α-treated groups. Data are means ± SD. *Significant difference compared with each time point control ( P

    Techniques Used: Western Blot

    Degradation of p105 and IκB by TNF-α treatment. HK-2 cells were treated with 20 ng/ml TNF-α for 0–90 min. After treatment, degradation of p105 ( A , n = 4) and IκBα ( B , n = 4) were evaluated by Western blot analysis. Degradation levels of p105 and p65 were normalized based on β-actin expression levels. Open columns, control groups; filled columns, TNF-α-treated groups. Data are means ± SD. *Significant difference compared with each time point control ( P
    Figure Legend Snippet: Degradation of p105 and IκB by TNF-α treatment. HK-2 cells were treated with 20 ng/ml TNF-α for 0–90 min. After treatment, degradation of p105 ( A , n = 4) and IκBα ( B , n = 4) were evaluated by Western blot analysis. Degradation levels of p105 and p65 were normalized based on β-actin expression levels. Open columns, control groups; filled columns, TNF-α-treated groups. Data are means ± SD. *Significant difference compared with each time point control ( P

    Techniques Used: Western Blot, Expressing

    Contributions of p50/p65 heterodimer and p50/p50 homodimer to suppression of AGT expression by TNF-α. HK-2 cells were treated with negative small interfering RNA (siRNA), p65-siRNA, and p105/p50-siRNA for 48 h. After treatment, expression levels of p65 ( A , left , n = 3), p105 ( A , center , n = 3), and p50 ( A , right , n = 3) were determined using Western blot analysis. NC, negative control group (absent of any siRNA treatment); NS, negative siRNA-treated group; Si, siRNA-treated group (p65-siRNA or p105/p50-siRNA). Data are expressed as means ± SD. In A : *significant difference compared with the corresponding NS group ( P
    Figure Legend Snippet: Contributions of p50/p65 heterodimer and p50/p50 homodimer to suppression of AGT expression by TNF-α. HK-2 cells were treated with negative small interfering RNA (siRNA), p65-siRNA, and p105/p50-siRNA for 48 h. After treatment, expression levels of p65 ( A , left , n = 3), p105 ( A , center , n = 3), and p50 ( A , right , n = 3) were determined using Western blot analysis. NC, negative control group (absent of any siRNA treatment); NS, negative siRNA-treated group; Si, siRNA-treated group (p65-siRNA or p105/p50-siRNA). Data are expressed as means ± SD. In A : *significant difference compared with the corresponding NS group ( P

    Techniques Used: Expressing, Small Interfering RNA, Western Blot, Negative Control

    18) Product Images from "Bacteroides fragilis Enterotoxin Induces Human ?-Defensin-2 Expression in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase/I?B Kinase/NF-?B-Dependent Pathway ▿"

    Article Title: Bacteroides fragilis Enterotoxin Induces Human ?-Defensin-2 Expression in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase/I?B Kinase/NF-?B-Dependent Pathway ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00118-10

    Suppression of IKK activity by siRNA in HT-29 intestinal epithelial cells stimulated with BFT. (A) HT-29 cells were stimulated with BFT (300 ng/ml) for the indicated periods. Phosphorylation and protein expression of IKK-α, IKK-β, and
    Figure Legend Snippet: Suppression of IKK activity by siRNA in HT-29 intestinal epithelial cells stimulated with BFT. (A) HT-29 cells were stimulated with BFT (300 ng/ml) for the indicated periods. Phosphorylation and protein expression of IKK-α, IKK-β, and

    Techniques Used: Activity Assay, Expressing

    19) Product Images from "Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis"

    Article Title: Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00230

    METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p
    Figure Legend Snippet: METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p

    Techniques Used: Expressing, Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    20) Product Images from "Unsaturated Fatty Acids Revert Diet-Induced Hypothalamic Inflammation in Obesity"

    Article Title: Unsaturated Fatty Acids Revert Diet-Induced Hypothalamic Inflammation in Obesity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030571

    GPR120 signal transduction in the hypothalamus. Five µm sections of the hypothalamus obtained from obese Wistar rats were labeled with anti-GPR120 (green) and NPY (red) antibodies, low (A) and high (B) magnifications are depicted. Icv cannulated obese Wistar rats were acutely treated with diluent (albumin, Alb), ω3 or ω9 fatty acids and then used in immunoprecipitation (IP)/immunoblotting (IB) experiments employing antibodies against GRP120 (C), β-arrestin 2 (C and D), TAK1 (E), and TAB1 (D and E). In all experiments n = 5. In C-E, *p
    Figure Legend Snippet: GPR120 signal transduction in the hypothalamus. Five µm sections of the hypothalamus obtained from obese Wistar rats were labeled with anti-GPR120 (green) and NPY (red) antibodies, low (A) and high (B) magnifications are depicted. Icv cannulated obese Wistar rats were acutely treated with diluent (albumin, Alb), ω3 or ω9 fatty acids and then used in immunoprecipitation (IP)/immunoblotting (IB) experiments employing antibodies against GRP120 (C), β-arrestin 2 (C and D), TAK1 (E), and TAB1 (D and E). In all experiments n = 5. In C-E, *p

    Techniques Used: Transduction, Labeling, Immunoprecipitation

    21) Product Images from "Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages"

    Article Title: Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages

    Journal: Foods

    doi: 10.3390/foods9030269

    Schematic model showing the inhibitory effects of compound 1 on lipopolysaccharide-induced inflammatory response in RAW 264.7 mouse macrophages through the inhibition of IKKα/β, I-κBα, NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the activities of IL-6 and TNF-α. 1,5-dimethyl citrate ( 1 ).
    Figure Legend Snippet: Schematic model showing the inhibitory effects of compound 1 on lipopolysaccharide-induced inflammatory response in RAW 264.7 mouse macrophages through the inhibition of IKKα/β, I-κBα, NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the activities of IL-6 and TNF-α. 1,5-dimethyl citrate ( 1 ).

    Techniques Used: Inhibition

    Effects of compound 1 on the LPS-induced expression of iNOS and COX-2 in RAW 264.7 mouse macrophages. ( A ) Representative Western blots of iNOS, COX-2, and GAPDH protein expression. Quantitative graph of ( B ) iNOS and ( C ) COX-2 (mean ± SD, * p
    Figure Legend Snippet: Effects of compound 1 on the LPS-induced expression of iNOS and COX-2 in RAW 264.7 mouse macrophages. ( A ) Representative Western blots of iNOS, COX-2, and GAPDH protein expression. Quantitative graph of ( B ) iNOS and ( C ) COX-2 (mean ± SD, * p

    Techniques Used: Expressing, Western Blot

    22) Product Images from "USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation"

    Article Title: USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2011.11

    Knockdown of USP4 expression enhances TNF α -induced TAK1 polyubiquitination and association of TNFR1 with TAK1 and IKK β . ( a ) Knockdown of USP4 expression enhances the TNF α -induced TAK1 polyubiquitination in the cells. USP4-knockdown
    Figure Legend Snippet: Knockdown of USP4 expression enhances TNF α -induced TAK1 polyubiquitination and association of TNFR1 with TAK1 and IKK β . ( a ) Knockdown of USP4 expression enhances the TNF α -induced TAK1 polyubiquitination in the cells. USP4-knockdown

    Techniques Used: Expressing

    23) Product Images from "Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis"

    Article Title: Role of CXCR1 and Interleukin-8 in Methamphetamine-Induced Neuronal Apoptosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00230

    METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p
    Figure Legend Snippet: METH increases the expression of chemokine interleukin (IL)-8 via nuclear translocation of NF-κB in astrocytes. U87MG cells were exposed to 0.5 mM, 1 mM, 1.5 mM, 2 mM and 2.5 mM of METH for 24 h (A) or 2 mM of METH for 24 h (D) . (F) U87MG cells were exposed to Bay 11–7082 (10 μM) for 12 h prior to METH (2 mM) treatment as indicated. Western blot (A,D,F) and quantitative analyses (B,E,G) were performed to determine IL-8, IKK-α, IKK-β, phospho-IKK-α/β, IκB, NF-κB and phospho-NF-κB protein expression. β-actin was used as a loading control. ELISA (C,H) was performed to detect the concentration of IL-8 in culture medium supernatant. Fold induction relative to the control group is shown. *Represents a significant difference as compared with the non-METH-treated group, * p

    Techniques Used: Expressing, Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    24) Product Images from "Cystatin E/M Suppresses Tumor Cell Growth through Cytoplasmic Retention of NF-κB"

    Article Title: Cystatin E/M Suppresses Tumor Cell Growth through Cytoplasmic Retention of NF-κB

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00878-15

    Inhibition of canonical NF-κB signaling pathway by cystatin E/M protein. (A) Basal expression of nonphosphorylated IκBα was seen in HeLa cells and was not altered after doxycycline treatment. However, IκBα expression increased in CST6 cells after doxycycline treatment, correlating to decreased phosphor lation of IκBα. Treatment with TNF-α enhanced phospho-IκBα expression in both the HeLa and CST6 cell lines, and the expression was not altered in HeLa cells after doxycycline treatment. On the other hand, treatment with doxycycline led to reduced expression of phospho-IκBα in CST6 cells. (B) The basal IKKβ expression level was high in the control CST6 cells, phospho-IKKβ expression was visualized only with the addition of TNF-α, and the expression was reduced in doxycycline-treated cells. (C) Induced cystatin E/M expression did not inhibit endogenous activation of p100 (NF-κB2) to produce p52 in CST6 cells. In HEK 293T cells, exogenous NIK expression led to activation of myc-tag p100 to produce myc-p52. While this activation was inhibited by cIAP1/2 expression, inhibition was not observed with the expression of cystatin E/M. (D) Mechanistic representation showing inhibition of the IKKβ-mediated canonical and not the NIK-mediated noncanonical NF-κB signaling pathway by cystatin E/M. TNFR, tumor necrosis factor receptor; TNFSFR, tumor necrosis factor superfamily receptor.
    Figure Legend Snippet: Inhibition of canonical NF-κB signaling pathway by cystatin E/M protein. (A) Basal expression of nonphosphorylated IκBα was seen in HeLa cells and was not altered after doxycycline treatment. However, IκBα expression increased in CST6 cells after doxycycline treatment, correlating to decreased phosphor lation of IκBα. Treatment with TNF-α enhanced phospho-IκBα expression in both the HeLa and CST6 cell lines, and the expression was not altered in HeLa cells after doxycycline treatment. On the other hand, treatment with doxycycline led to reduced expression of phospho-IκBα in CST6 cells. (B) The basal IKKβ expression level was high in the control CST6 cells, phospho-IKKβ expression was visualized only with the addition of TNF-α, and the expression was reduced in doxycycline-treated cells. (C) Induced cystatin E/M expression did not inhibit endogenous activation of p100 (NF-κB2) to produce p52 in CST6 cells. In HEK 293T cells, exogenous NIK expression led to activation of myc-tag p100 to produce myc-p52. While this activation was inhibited by cIAP1/2 expression, inhibition was not observed with the expression of cystatin E/M. (D) Mechanistic representation showing inhibition of the IKKβ-mediated canonical and not the NIK-mediated noncanonical NF-κB signaling pathway by cystatin E/M. TNFR, tumor necrosis factor receptor; TNFSFR, tumor necrosis factor superfamily receptor.

    Techniques Used: Inhibition, Expressing, Activation Assay

    25) Product Images from "Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages"

    Article Title: Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages

    Journal: Foods

    doi: 10.3390/foods9030269

    Schematic model showing the inhibitory effects of compound 1 on lipopolysaccharide-induced inflammatory response in RAW 264.7 mouse macrophages through the inhibition of IKKα/β, I-κBα, NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the activities of IL-6 and TNF-α. 1,5-dimethyl citrate ( 1 ).
    Figure Legend Snippet: Schematic model showing the inhibitory effects of compound 1 on lipopolysaccharide-induced inflammatory response in RAW 264.7 mouse macrophages through the inhibition of IKKα/β, I-κBα, NF-κB p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the activities of IL-6 and TNF-α. 1,5-dimethyl citrate ( 1 ).

    Techniques Used: Inhibition

    Effects of compound 1 on the LPS-induced expression of the IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), and NF-κB p65 proteins in RAW 264.7 mouse macrophages. ( A ) Representative Western blots of IKKα/β, I-κBα, NF-κB p65, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression. Quantitative graph of ( B ) p-IKKα/β, ( C ) p-I-κBα, and ( D ) p-p65 (mean ± SD, * p
    Figure Legend Snippet: Effects of compound 1 on the LPS-induced expression of the IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), and NF-κB p65 proteins in RAW 264.7 mouse macrophages. ( A ) Representative Western blots of IKKα/β, I-κBα, NF-κB p65, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein expression. Quantitative graph of ( B ) p-IKKα/β, ( C ) p-I-κBα, and ( D ) p-p65 (mean ± SD, * p

    Techniques Used: Expressing, Western Blot

    26) Product Images from "Interleukin-1-induced NF-κB Activation Is NEMO-dependent but Does Not Require IKKβ"

    Article Title: Interleukin-1-induced NF-κB Activation Is NEMO-dependent but Does Not Require IKKβ

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M609613200

    IL-1 activates NEMO-dependent classical NF- κ B in IKK β −/− MEFs A , WT, NEMO-deficient, IKK α −/− , and IKK β −/− MEFs were either untreated or incubated with IL-1 for the times indicated, and then nuclear extracts were prepared and used for EMSA. Assays were performed using either a consensus NF- κ B binding site probe ( upper panel ) or an Oct1 probe as a loading control ( lower panel ). B , the MEFs shown ( top ) were either untreated or incubated with IL-1 for 30 min, and then nuclear lysates were prepared. For supershift analysis, samples were incubated prior to the EMSA reaction either in the absence of antibodies (−) or with anti-p65 or -p50 as shown. The positions of the shifted NF- κ B complex (*) and supershifted p65- and p50-containing complexes are indicated ( right ). C , WT and IKK β −/− MEFs were either untreated (−) or stimulated with anti-LT β R ( LT ) or IL-1 (10 ng/ml) for 8 h, and then lysates were immunoblotted and probed with either anti-p100/p52 ( upper panel ) or anti-tubulin ( lower panel ). D , MEFs were transiently transfected with the NF- κ B-dependent reporter pBIIx-firefly luciferase together with β -actin Renilla luciferase. Twenty-four hours later, the cells were either untreated or incubated for a further 5 h with IL-1 (10 ng/ml), and then NF- κ B activity was determined by dual luciferase assay. The data are expressed for each MEF line as fold values relative to the basal activity in untreated cells that was normalized between the cell lines ( dotted line ).
    Figure Legend Snippet: IL-1 activates NEMO-dependent classical NF- κ B in IKK β −/− MEFs A , WT, NEMO-deficient, IKK α −/− , and IKK β −/− MEFs were either untreated or incubated with IL-1 for the times indicated, and then nuclear extracts were prepared and used for EMSA. Assays were performed using either a consensus NF- κ B binding site probe ( upper panel ) or an Oct1 probe as a loading control ( lower panel ). B , the MEFs shown ( top ) were either untreated or incubated with IL-1 for 30 min, and then nuclear lysates were prepared. For supershift analysis, samples were incubated prior to the EMSA reaction either in the absence of antibodies (−) or with anti-p65 or -p50 as shown. The positions of the shifted NF- κ B complex (*) and supershifted p65- and p50-containing complexes are indicated ( right ). C , WT and IKK β −/− MEFs were either untreated (−) or stimulated with anti-LT β R ( LT ) or IL-1 (10 ng/ml) for 8 h, and then lysates were immunoblotted and probed with either anti-p100/p52 ( upper panel ) or anti-tubulin ( lower panel ). D , MEFs were transiently transfected with the NF- κ B-dependent reporter pBIIx-firefly luciferase together with β -actin Renilla luciferase. Twenty-four hours later, the cells were either untreated or incubated for a further 5 h with IL-1 (10 ng/ml), and then NF- κ B activity was determined by dual luciferase assay. The data are expressed for each MEF line as fold values relative to the basal activity in untreated cells that was normalized between the cell lines ( dotted line ).

    Techniques Used: Incubation, Binding Assay, Transfection, Luciferase, Activity Assay

    Dominant negative IKK β (K44M) blocks TNF- but not IL-1-induced I κ B α degradation A , WT MEFs (MEFs 1) were either mock-transduced ( Control, left panel ) or stably transduced with LZRS −EGFP ( right panel ), and the percentage of EGFP-positive cells was determined by FACS analysis. B and C , stably transduced WT LZRS −EGFP or LZRS −IKK β (K44M) MEFs were treated for the times indicated with TNF ( B ) or IL-1 ( C ), and then lysates were immunoblotted using antibodies against I κ B α , IKK β , and α -tubulin as shown.
    Figure Legend Snippet: Dominant negative IKK β (K44M) blocks TNF- but not IL-1-induced I κ B α degradation A , WT MEFs (MEFs 1) were either mock-transduced ( Control, left panel ) or stably transduced with LZRS −EGFP ( right panel ), and the percentage of EGFP-positive cells was determined by FACS analysis. B and C , stably transduced WT LZRS −EGFP or LZRS −IKK β (K44M) MEFs were treated for the times indicated with TNF ( B ) or IL-1 ( C ), and then lysates were immunoblotted using antibodies against I κ B α , IKK β , and α -tubulin as shown.

    Techniques Used: Dominant Negative Mutation, Stable Transfection, Transduction, FACS

    Distinct modes of IKK complex activity transduce TNF and IL-1 signaling to NF- κ B Our findings verify that TNF-induced I κ B α degradation and classical NF- κ B activation is critically dependent upon NEMO and IKK β ( left ). Similarly, IL-1 signaling absolutely requires intact NEMO ( right ). However, IL-1-induced I κ B α degradation, NF- κ B nuclear translocation, and NF- κ B transcriptional activity occurs in the absence of IKK β , demonstrating that NEMO and IKK α can form a signaling complex capable of activating the classical NF- κ B pathway in response to certain pro-inflammatory stimuli.
    Figure Legend Snippet: Distinct modes of IKK complex activity transduce TNF and IL-1 signaling to NF- κ B Our findings verify that TNF-induced I κ B α degradation and classical NF- κ B activation is critically dependent upon NEMO and IKK β ( left ). Similarly, IL-1 signaling absolutely requires intact NEMO ( right ). However, IL-1-induced I κ B α degradation, NF- κ B nuclear translocation, and NF- κ B transcriptional activity occurs in the absence of IKK β , demonstrating that NEMO and IKK α can form a signaling complex capable of activating the classical NF- κ B pathway in response to certain pro-inflammatory stimuli.

    Techniques Used: Activity Assay, Activation Assay, Translocation Assay

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    Article Title: Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction *
    Article Snippet: .. The following antibodies were used: TRAF3IP2 (1:500; #NB100-56740, Novus), ANP (1:400; #NBP2-14873, Novus), IL-6 (1:200; #AF-406-NA, R & D Systems), α-tubulin (1:1000; #2144, Cell Signaling Technology or CST), phospho-p65 (Ser536 ; 1:1000; #3031, CST), p65 (1:1000; #8242, CST), phospho-c-Jun (Ser63 , 1:1000; #9261, CST), c-Jun (1:1000; #9165, CST), JNK (1:1000; #9252, CST), phospho-JNK (Thr183 /Tyr185 ; 1: 1000, #4668, CST), IKKβ (1:1000; #2678, CST), and phospho-IKKα/β (Ser176 /180 ; 1:1000; #2694, CST), LOX (1:500; #sc-373995, Santa Cruz Biotechnology or SCB), IL-18 (1:100; #sc-7954, SCB), BNP (1:400, #sc-67455, SCB), ColIα1 (1:2000; #ab34170, Abcam), ColIIIα1 (1:2000; #ab7778, Abcam), CTGF (1:500; #210303, United States Biological), and β-MyHC (1:400, #M9850-11C, United States Biological). .. Data were analyzed using Microsoft Excel, Clampfit (Molecular Device, Sunnyvale, CA), and Origin 7 (OriginLab Corp., Northampton, MA) programs.

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    Article Title: Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline
    Article Snippet: Antibodies specific for AKT (9272), Bcl-xL (2762), CUL-4A (2699), ERK (4695), HSP90β (5087), JAK1 (3344), JAK2 (3230), c-Myc (9402), NEDD8 (polyclonal, 2745), NEDD8 (monoclonal, 2754), nucleolin (12247), phospho-ERK (9101), phospho-IκBα (9241), phospho-IKKα/β (2694), phospho-STAT3 (Tyr705, 9145), phospho-STAT3 (Ser727, 9134), STAT3a (8768), and STAT3 (9139) were from Cell Signaling Technology.

    Article Title: Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A
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    Cell Signaling Technology Inc rabbit polyclonal anti phospho iκb kinase ikk α β antibody
    Rabbit Polyclonal Anti Phospho Iκb Kinase Ikk α β Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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