phospho ikkα β  (Cell Signaling Technology Inc)

 
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    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
    Buy from Supplier


    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
    Average 91 stars, based on 9254 article reviews
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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 "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

    3) 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

    4) Product Images from "TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival"

    Article Title: TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2008.06.006

    Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.
    Figure Legend Snippet: Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.

    Techniques Used: Activation Assay

    5) Product Images from "Age-dependent regulation of antioxidant genes by p38α MAPK in the liver"

    Article Title: Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.02.017

    Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.
    Figure Legend Snippet: Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.

    Techniques Used: Western Blot, Mouse Assay

    6) Product Images from "Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction *"

    Article Title: Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.724138

    Characterization of TRAF3IP2-Tg mice. A and B , TRAF3IP2 mRNA ( A ) and protein expression ( B ) in LV tissues of 2-month-old male Tg and NTg mice analyzed by RT-qPCR and immunoblotting. Each lane in panel B represents an individual animal. Densitometric analysis of immunoreactive bands in panel B is summarized on the right. C and D , tissue specificity of transgene-derived TRAF3IP2 overexpression was analyzed by immunoblotting ( N , NTg; T , Tg). Densitometric analysis of immunoreactive bands is summarized in D. E , immunofluorescence analysis of TRAF3IP2 overexpression in the heart co-localized with WGA. Scale : 50 μm. TRAF3IP2 overexpression is localized predominantly to cardiomyocytes ( CM ) but not endothelial or smooth muscle cells of blood vessels ( BV ). Intensity of TRAF3IP2 immunofluorescence signals were quantified and summarized on the right. F and G , TRAF3IP2 overexpression induces activation of IKKβ, p65, JNK, c-Jun, c/EBPβ, and p38 MAPK in LV tissues from male. Densitometric analysis of immune-reactive bands is summarized on the right ( F ) but not female Tg ( G ) as analyzed by immunoblotting using activation-specific antibodies. n = 4/group, Error bars represent S.E. *, p
    Figure Legend Snippet: Characterization of TRAF3IP2-Tg mice. A and B , TRAF3IP2 mRNA ( A ) and protein expression ( B ) in LV tissues of 2-month-old male Tg and NTg mice analyzed by RT-qPCR and immunoblotting. Each lane in panel B represents an individual animal. Densitometric analysis of immunoreactive bands in panel B is summarized on the right. C and D , tissue specificity of transgene-derived TRAF3IP2 overexpression was analyzed by immunoblotting ( N , NTg; T , Tg). Densitometric analysis of immunoreactive bands is summarized in D. E , immunofluorescence analysis of TRAF3IP2 overexpression in the heart co-localized with WGA. Scale : 50 μm. TRAF3IP2 overexpression is localized predominantly to cardiomyocytes ( CM ) but not endothelial or smooth muscle cells of blood vessels ( BV ). Intensity of TRAF3IP2 immunofluorescence signals were quantified and summarized on the right. F and G , TRAF3IP2 overexpression induces activation of IKKβ, p65, JNK, c-Jun, c/EBPβ, and p38 MAPK in LV tissues from male. Densitometric analysis of immune-reactive bands is summarized on the right ( F ) but not female Tg ( G ) as analyzed by immunoblotting using activation-specific antibodies. n = 4/group, Error bars represent S.E. *, p

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Derivative Assay, Over Expression, Immunofluorescence, Whole Genome Amplification, Activation Assay

    7) Product Images from "Signal transduction mechanisms involved in S100A4-induced activation of the transcription factor NF-?B"

    Article Title: Signal transduction mechanisms involved in S100A4-induced activation of the transcription factor NF-?B

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-241

    A. S100A4 induces phosphorylation of IKKα/β . Western blot of immunoprecipitated IKK complex from II-11b cells treated with 2 μM S100A4 for the indicated time periods and analyzed for expression of phosphorylated IKKα/β. IKKα was used as loading control. B. H-7 and staurosporine inhibit IKK-mediated IκBα phosphorylation in vitro. Upper panel: Kinase assay showing phosphorylation of recombinant IκBα by immunoprecipitated IKK complex from unstimulated II-11b cells or cells stimulated with 2 μM S100A4 for 15 minutes, with or without H-7 (30 μM) or staurosporine (0.4 μM) present in the reaction mixture. Lower panel: Western blot showing IKKα as a loading control. C. Densitometric quantification of the signals shown in Fig. 5B. The bars show IκBα phosphorylation relative to IKKα expression. One of three independent experiments is quantified due to high background in the other experiments. D. Treatment with H-7 or staurosporine did not influence the level of phosphorylated IKKα/β upon S100A4-stimulation for 15 minutes. Immunoprecipitated IKK complex was analyzed by immunoblotting using anti-phospho IKKα/β antibody. IKKα is used as loading control. E. Densitometric quantification of Fig. 5D. Bars show phosphorylated IKKα/β relative to IKKα expression. B and D are representative results of three independent experiments. St = staurosporine.
    Figure Legend Snippet: A. S100A4 induces phosphorylation of IKKα/β . Western blot of immunoprecipitated IKK complex from II-11b cells treated with 2 μM S100A4 for the indicated time periods and analyzed for expression of phosphorylated IKKα/β. IKKα was used as loading control. B. H-7 and staurosporine inhibit IKK-mediated IκBα phosphorylation in vitro. Upper panel: Kinase assay showing phosphorylation of recombinant IκBα by immunoprecipitated IKK complex from unstimulated II-11b cells or cells stimulated with 2 μM S100A4 for 15 minutes, with or without H-7 (30 μM) or staurosporine (0.4 μM) present in the reaction mixture. Lower panel: Western blot showing IKKα as a loading control. C. Densitometric quantification of the signals shown in Fig. 5B. The bars show IκBα phosphorylation relative to IKKα expression. One of three independent experiments is quantified due to high background in the other experiments. D. Treatment with H-7 or staurosporine did not influence the level of phosphorylated IKKα/β upon S100A4-stimulation for 15 minutes. Immunoprecipitated IKK complex was analyzed by immunoblotting using anti-phospho IKKα/β antibody. IKKα is used as loading control. E. Densitometric quantification of Fig. 5D. Bars show phosphorylated IKKα/β relative to IKKα expression. B and D are representative results of three independent experiments. St = staurosporine.

    Techniques Used: Western Blot, Immunoprecipitation, Expressing, In Vitro, Kinase Assay, Recombinant

    8) Product Images from "cIAP2 represses IKK?/?-mediated activation of MDM2 to prevent p53 degradation"

    Article Title: cIAP2 represses IKK?/?-mediated activation of MDM2 to prevent p53 degradation

    Journal: Cell Cycle

    doi: 10.4161/cc.22223

    Figure 6. Hypothetical model of cIAP2-dependent regulation of p53. cIAP2 reduction results in the phosphorylation of IKKα which then activates IKKβ resulting in canonical NFκB activity. NFκB promotes a transient
    Figure Legend Snippet: Figure 6. Hypothetical model of cIAP2-dependent regulation of p53. cIAP2 reduction results in the phosphorylation of IKKα which then activates IKKβ resulting in canonical NFκB activity. NFκB promotes a transient

    Techniques Used: Activity Assay

    Figure 3. IKKα and IKKβ are both required for MDM2 activation and p53 downregulation following cIAP2 knockdown. MCF-10AT1 cells were transfected with cIAP2 or IKKα siRNA then infected with an adenovirus expressing a dominant-negative
    Figure Legend Snippet: Figure 3. IKKα and IKKβ are both required for MDM2 activation and p53 downregulation following cIAP2 knockdown. MCF-10AT1 cells were transfected with cIAP2 or IKKα siRNA then infected with an adenovirus expressing a dominant-negative

    Techniques Used: Activation Assay, Transfection, Infection, Expressing, Dominant Negative Mutation

    9) Product Images from "Age-dependent regulation of antioxidant genes by p38α MAPK in the liver"

    Article Title: Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.02.017

    Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.
    Figure Legend Snippet: Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.

    Techniques Used: Western Blot, Mouse Assay

    10) Product Images from "Age-dependent regulation of antioxidant genes by p38α MAPK in the liver"

    Article Title: Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.02.017

    Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.
    Figure Legend Snippet: Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.

    Techniques Used: Western Blot, Mouse Assay

    11) Product Images from "TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival"

    Article Title: TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2008.06.006

    Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.
    Figure Legend Snippet: Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.

    Techniques Used: Activation Assay

    12) Product Images from "Grape Seed Extract Attenuates Hepatitis C Virus Replication and Virus-Induced Inflammation"

    Article Title: Grape Seed Extract Attenuates Hepatitis C Virus Replication and Virus-Induced Inflammation

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2016.00490

    Grape seed extract reduced NF-κB transactivity and MAPK phosphorylation for suppression of COX-2 expression in HCV replicon cells. (A) GSE reduced NF-κB transactivity in Ava5 cells. Ava5 cells were transiently transfected with pNF-κB-Luc, which contained an NF-κB binding element linked firefly luciferase reporter gene. The pNF-κB-Luc-transfected cells were treated with 20 μg/ml of GSE for 3 days. Subsequently, the extracted lysates of transfected cells were analyzed by luciferase activity assay. The relative NF-κB transactivity was presented as fold changes compared to parental Huh-7 cells in which luciferase activity was presented as 1. The GSE treatment downregulated (B) NF-κB phosphorylation and (C) the HCV-induced NF-κB signaling pathway. Ava5 cells were treated with GSE in different concentrations (0–20 μg/ml) for 3 days and the nuclear lysates were isolated as described in Section “Materials and Methods.” The nuclear translocation of NF-κB were analyzed by Western blotting with anti-phospho-p65 and anti-Lamin B (loading control) antibodies. The effects of GSE on NF-κB regulation were analyzed by Western blotting with various antibodies against IKKα, phospho-IKKα/β, IκB-α, phospho-IκB-α, and GAPDH (loading control). (D) GSE treatment reduced the phosphorylation level of ERK and JNK. Ava5 cells were treated with 20 μg/ml of GSE and the lysates extracted at the indicated time points after the treatment. The protein expressions were analyzed by Western blotting with antibodies against MAPK (ERK1/2, p38, and JNK), phospho-MAPK (p-ERK1/2, p-p38, and p-JNK), and GAPDH (loading control). Data are represented as the mean ± SD for three independent experiments. ∗ P
    Figure Legend Snippet: Grape seed extract reduced NF-κB transactivity and MAPK phosphorylation for suppression of COX-2 expression in HCV replicon cells. (A) GSE reduced NF-κB transactivity in Ava5 cells. Ava5 cells were transiently transfected with pNF-κB-Luc, which contained an NF-κB binding element linked firefly luciferase reporter gene. The pNF-κB-Luc-transfected cells were treated with 20 μg/ml of GSE for 3 days. Subsequently, the extracted lysates of transfected cells were analyzed by luciferase activity assay. The relative NF-κB transactivity was presented as fold changes compared to parental Huh-7 cells in which luciferase activity was presented as 1. The GSE treatment downregulated (B) NF-κB phosphorylation and (C) the HCV-induced NF-κB signaling pathway. Ava5 cells were treated with GSE in different concentrations (0–20 μg/ml) for 3 days and the nuclear lysates were isolated as described in Section “Materials and Methods.” The nuclear translocation of NF-κB were analyzed by Western blotting with anti-phospho-p65 and anti-Lamin B (loading control) antibodies. The effects of GSE on NF-κB regulation were analyzed by Western blotting with various antibodies against IKKα, phospho-IKKα/β, IκB-α, phospho-IκB-α, and GAPDH (loading control). (D) GSE treatment reduced the phosphorylation level of ERK and JNK. Ava5 cells were treated with 20 μg/ml of GSE and the lysates extracted at the indicated time points after the treatment. The protein expressions were analyzed by Western blotting with antibodies against MAPK (ERK1/2, p38, and JNK), phospho-MAPK (p-ERK1/2, p-p38, and p-JNK), and GAPDH (loading control). Data are represented as the mean ± SD for three independent experiments. ∗ P

    Techniques Used: Expressing, Transfection, Binding Assay, Luciferase, Activity Assay, Isolation, Translocation Assay, Western Blot

    13) Product Images from "TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival"

    Article Title: TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2008.06.006

    Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.
    Figure Legend Snippet: Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.

    Techniques Used: Activation Assay

    14) Product Images from "Inhibition of NF-?B activity by HIV-1 Vpr is dependent on Vpr binding protein"

    Article Title: Inhibition of NF-?B activity by HIV-1 Vpr is dependent on Vpr binding protein

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.24226

    Vpr mediated regulation of IκBα and NF-κB p65 signaling is altered by HSP27 and VprBP knockdown
    Figure Legend Snippet: Vpr mediated regulation of IκBα and NF-κB p65 signaling is altered by HSP27 and VprBP knockdown

    Techniques Used:

    15) Product Images from "Hepatitis B Virus Polymerase Suppresses NF-?B Signaling by Inhibiting the Activity of IKKs via Interaction with Hsp90?"

    Article Title: Hepatitis B Virus Polymerase Suppresses NF-?B Signaling by Inhibiting the Activity of IKKs via Interaction with Hsp90?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091658

    HBV Pol inhibits the activities of IKKα and IKKβ and the degradation of IκBα protein. 293( A .) and HepG2 cells ( B .) were transfected with the plasmids as indicated. Cells were examined at 24 h posttransfection and after stimulation with TNF-α (10 ng/ml) or not for 15 min. Cell extracts were prepared, and IκBα, p-IKKα and p-IKKβ levels were analyzed by western blots. C . 293 T cells were transfected with the HBV Pol or the empty vector for 24 h, then the proteasome inhibitor, MG-132 (20 µM), was added for the indicated times, and cell extracts were prepared and assayed with a phosphoserine IκBα (P-IκBα) antibody. D . The cell extracts were immunoprecipitated by anti-IKKα or anti-IKKβ antibodies and further analyzed by an in vitro kinase assay (KA) using GST-IκBα (1–54) as the IKK substrate. The IKK protein level in each precipitate was also determined by western blot. β-actin was used as the control for the western blots.
    Figure Legend Snippet: HBV Pol inhibits the activities of IKKα and IKKβ and the degradation of IκBα protein. 293( A .) and HepG2 cells ( B .) were transfected with the plasmids as indicated. Cells were examined at 24 h posttransfection and after stimulation with TNF-α (10 ng/ml) or not for 15 min. Cell extracts were prepared, and IκBα, p-IKKα and p-IKKβ levels were analyzed by western blots. C . 293 T cells were transfected with the HBV Pol or the empty vector for 24 h, then the proteasome inhibitor, MG-132 (20 µM), was added for the indicated times, and cell extracts were prepared and assayed with a phosphoserine IκBα (P-IκBα) antibody. D . The cell extracts were immunoprecipitated by anti-IKKα or anti-IKKβ antibodies and further analyzed by an in vitro kinase assay (KA) using GST-IκBα (1–54) as the IKK substrate. The IKK protein level in each precipitate was also determined by western blot. β-actin was used as the control for the western blots.

    Techniques Used: Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation, In Vitro, Kinase Assay

    HBV Pol suppresses NF-κB activity in HEK 293 T, HepG2, and Huh7 cells. A . Western blot analysis revealed the expression of the HBV Pol in HEK 293 T (lanes 1 and 2), HepG2 (lanes 3 and 4), and Huh7 (lanes 5 and 6) cells. Cells were transfected with 0.8 µg of pAHC-Pol expressing the HBV Pol (lanes 1, 3, and 5) or 0.8 µg of empty vector (pAHC) as a negative control (lanes 2, 4, and 6). At 32 h posttransfection, expression of the plasmids was determined by western blot using a mouse anti-HA antibody. B . HepG2 and Huh7 cells were cotransfected with 0.1 µg of pNF-κB-Luc (5×NF-κB binding site promoter-driven luciferase reporter plasmid) and 10 ng of pTK-Renilla-Luc along with 0.6 µg of pAHC-Pol or 0.6 µg of pAHC-X as a positive control and normalized to the pAHC empty vector. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. C . 293 T cells were cotransfected with 0.1 µg of 5×pNF-κB-Luc and 25 µg of pTK-Renilla-Luc along with different amounts of pAHC-Pol (lane 3, 0 ng; lane 4, 200 ng; lane 5, 400 ng; lane 6, 600 ng). The total amount of plasmid was adjusted with the empty vector (pAHC) to 600 ng. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. D . HepG2 cells were cotransfected with 100 ng of 5×pNF-κB-Luc and 25 ng of pTK-Renilla-Luc together with 100 ng of the activating plasmid, pRK-X (where X is TRAF2, TRAF6, IKKα, or IKKβ), or the corresponding empty vector, pRK, along with 600 ng of pAHC-Pol or 600 ng of the pAHC empty vector as indicated below the graph. After 24 h of transfection, relative luciferase activities were determined. E . HepG2 cells were transfected with empty vector, WT, X-null, E-null, E/X-null and P-null HBV replicon together with the 5×pNF-κB-Luc reporter construct and pTK-Renilla-Luc, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. F . HepG2.2.15 and HepG2 cells were transfected with 100 ng of 5×pNF-κB-Luc and 25 ng of pTK-Renilla-Luc. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. G . HepG2 cells were transfected with increasing doses of the HBV Pol expression construct, along with the IFNβ luciferase reporter construct and TLR3 expression construct. Cells were stimulated with 25 mg/mL poly I:C directly added to the medium for 8 h. Luciferase activities correspond to the average of results from at least three independent experiments, and data are shown as means ± SE (**p
    Figure Legend Snippet: HBV Pol suppresses NF-κB activity in HEK 293 T, HepG2, and Huh7 cells. A . Western blot analysis revealed the expression of the HBV Pol in HEK 293 T (lanes 1 and 2), HepG2 (lanes 3 and 4), and Huh7 (lanes 5 and 6) cells. Cells were transfected with 0.8 µg of pAHC-Pol expressing the HBV Pol (lanes 1, 3, and 5) or 0.8 µg of empty vector (pAHC) as a negative control (lanes 2, 4, and 6). At 32 h posttransfection, expression of the plasmids was determined by western blot using a mouse anti-HA antibody. B . HepG2 and Huh7 cells were cotransfected with 0.1 µg of pNF-κB-Luc (5×NF-κB binding site promoter-driven luciferase reporter plasmid) and 10 ng of pTK-Renilla-Luc along with 0.6 µg of pAHC-Pol or 0.6 µg of pAHC-X as a positive control and normalized to the pAHC empty vector. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. C . 293 T cells were cotransfected with 0.1 µg of 5×pNF-κB-Luc and 25 µg of pTK-Renilla-Luc along with different amounts of pAHC-Pol (lane 3, 0 ng; lane 4, 200 ng; lane 5, 400 ng; lane 6, 600 ng). The total amount of plasmid was adjusted with the empty vector (pAHC) to 600 ng. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. D . HepG2 cells were cotransfected with 100 ng of 5×pNF-κB-Luc and 25 ng of pTK-Renilla-Luc together with 100 ng of the activating plasmid, pRK-X (where X is TRAF2, TRAF6, IKKα, or IKKβ), or the corresponding empty vector, pRK, along with 600 ng of pAHC-Pol or 600 ng of the pAHC empty vector as indicated below the graph. After 24 h of transfection, relative luciferase activities were determined. E . HepG2 cells were transfected with empty vector, WT, X-null, E-null, E/X-null and P-null HBV replicon together with the 5×pNF-κB-Luc reporter construct and pTK-Renilla-Luc, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. F . HepG2.2.15 and HepG2 cells were transfected with 100 ng of 5×pNF-κB-Luc and 25 ng of pTK-Renilla-Luc. At 24 h posttransfection, cells were treated with TNF-α (10 ng/ml) or left untreated for 1 h as indicated and then harvested for analysis by luciferase assay. G . HepG2 cells were transfected with increasing doses of the HBV Pol expression construct, along with the IFNβ luciferase reporter construct and TLR3 expression construct. Cells were stimulated with 25 mg/mL poly I:C directly added to the medium for 8 h. Luciferase activities correspond to the average of results from at least three independent experiments, and data are shown as means ± SE (**p

    Techniques Used: Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Binding Assay, Luciferase, Positive Control, Construct

    16) 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

    17) Product Images from "LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration"

    Article Title: LILRB4 signaling in leukemia cells mediates T cell suppression and tumor infiltration

    Journal: Nature

    doi: 10.1038/s41586-018-0615-z

    LILRB4-mediated intracellular signaling controls AML cell migration and T cell suppression. a, Expression and phosphorylation of three phosphatases in wild-type and lilrb4- KO THP-1 cells. b, Primary T cells and irradiated indicated THP-1 cells were cultured in the lower and upper chambers respectively. T cells were analyzed by flow cytometry after 7 days. n=4 biologically independent samples. c-d, Knockout of shp-2 reduces THP-1 cell short-term (20 hrs) and long-term (21 days) infiltration in NSG mice (n=5 mice). e, Upstream transcription factor analysis of RNA-seq data generated from lilrb4 -KO and WT THP-1 cells (n=2 biologically independent samples). Yellow dots highlighted the transcription factors involved in JAK/STATs and NF-κB pathways. f, Decreased phosphorylation of IKKα/β in lilrb4 -KO THP-1 cells. g, Decreased NFκB in the nuclear fraction in lilrb4 -KO THP-1 cells. h-i, The NF-κB inhibitor reversed T cell suppression by THP-1 cells ( h ) and decreased infiltration of MV4–11 cells ( i ) in an LILRB4-dependent manner (n=4 biologically independent samples). j, T cells isolated from healthy donors were supplemented with 25% condition medium (CM) of WT or lilrb4 -KO THP-1 cells. Representative cells were photographed (scale bar, 100 μm) and T cells were analyzed by flow cytometry (n=4 biologically independent samples). k-l, T cells were incubated with irradiated indicated THP-1 cells supplemented with indicated concentration of recombinant uPAR ( k ) or ARG-1 ( l ) proteins for 7 days and were analyzed by flow cytometry (n=4 biologically independent samples). m, Overexpression of uPAR ( plaur ) or ARG1 rescued infiltration defect of lilrb4 -KO MV4–11 cells (n=5 mice). ( a, f-g, j ) These experiments were repeated independently three times with similar results. See Methods for definition of box plot elements in ( b-d, h-m ). All p values were from two-tailed student t -test.
    Figure Legend Snippet: LILRB4-mediated intracellular signaling controls AML cell migration and T cell suppression. a, Expression and phosphorylation of three phosphatases in wild-type and lilrb4- KO THP-1 cells. b, Primary T cells and irradiated indicated THP-1 cells were cultured in the lower and upper chambers respectively. T cells were analyzed by flow cytometry after 7 days. n=4 biologically independent samples. c-d, Knockout of shp-2 reduces THP-1 cell short-term (20 hrs) and long-term (21 days) infiltration in NSG mice (n=5 mice). e, Upstream transcription factor analysis of RNA-seq data generated from lilrb4 -KO and WT THP-1 cells (n=2 biologically independent samples). Yellow dots highlighted the transcription factors involved in JAK/STATs and NF-κB pathways. f, Decreased phosphorylation of IKKα/β in lilrb4 -KO THP-1 cells. g, Decreased NFκB in the nuclear fraction in lilrb4 -KO THP-1 cells. h-i, The NF-κB inhibitor reversed T cell suppression by THP-1 cells ( h ) and decreased infiltration of MV4–11 cells ( i ) in an LILRB4-dependent manner (n=4 biologically independent samples). j, T cells isolated from healthy donors were supplemented with 25% condition medium (CM) of WT or lilrb4 -KO THP-1 cells. Representative cells were photographed (scale bar, 100 μm) and T cells were analyzed by flow cytometry (n=4 biologically independent samples). k-l, T cells were incubated with irradiated indicated THP-1 cells supplemented with indicated concentration of recombinant uPAR ( k ) or ARG-1 ( l ) proteins for 7 days and were analyzed by flow cytometry (n=4 biologically independent samples). m, Overexpression of uPAR ( plaur ) or ARG1 rescued infiltration defect of lilrb4 -KO MV4–11 cells (n=5 mice). ( a, f-g, j ) These experiments were repeated independently three times with similar results. See Methods for definition of box plot elements in ( b-d, h-m ). All p values were from two-tailed student t -test.

    Techniques Used: Migration, Expressing, Irradiation, Cell Culture, Flow Cytometry, Cytometry, Knock-Out, Mouse Assay, RNA Sequencing Assay, Generated, Isolation, Incubation, Concentration Assay, Recombinant, Over Expression, Two Tailed Test

    Detection of SHP-2/NF-κB signaling and uPAR and Arginase-1 expression in primary human monocytic AML cells. a, LILRB4-positive or -high CD33 + AML cells (red box) and LILRB4-negative or –low CD33 + AML cells (blue box) were gated for further intracellular staining of phosphorylated-SHP-2 at Y580, phosphorylated-IKKα/β at S176/S180, phosphorylated-NF-κB at S529, uPAR, and Arginase-1 (ARG1). Isotype IgG was used as negative controls. Red numbers indicate MFIs (mean fluorescence intensity) of LILRB4-positive or -high CD33 + AML cells; blue numbers indicate MFIs of LILRB4-negative or –low CD33 + AML cells. This experiment was repeated with 8 individual patient samples with similar results. b, Quantification of individual staining in LILRB4-positive or -high CD33 + AML cells versus in LILRB4-negative or low CD33 + AML cells. n=8 independent patients and see Methods for definition of box plot elements. p values were from two-tailed student t -test. c, Schematic for the mechanisms by which LILRB4 suppresses T cells and promotes leukemia infiltration.
    Figure Legend Snippet: Detection of SHP-2/NF-κB signaling and uPAR and Arginase-1 expression in primary human monocytic AML cells. a, LILRB4-positive or -high CD33 + AML cells (red box) and LILRB4-negative or –low CD33 + AML cells (blue box) were gated for further intracellular staining of phosphorylated-SHP-2 at Y580, phosphorylated-IKKα/β at S176/S180, phosphorylated-NF-κB at S529, uPAR, and Arginase-1 (ARG1). Isotype IgG was used as negative controls. Red numbers indicate MFIs (mean fluorescence intensity) of LILRB4-positive or -high CD33 + AML cells; blue numbers indicate MFIs of LILRB4-negative or –low CD33 + AML cells. This experiment was repeated with 8 individual patient samples with similar results. b, Quantification of individual staining in LILRB4-positive or -high CD33 + AML cells versus in LILRB4-negative or low CD33 + AML cells. n=8 independent patients and see Methods for definition of box plot elements. p values were from two-tailed student t -test. c, Schematic for the mechanisms by which LILRB4 suppresses T cells and promotes leukemia infiltration.

    Techniques Used: Expressing, Staining, Fluorescence, Two Tailed Test

    18) Product Images from "Age-dependent regulation of antioxidant genes by p38α MAPK in the liver"

    Article Title: Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.02.017

    Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.
    Figure Legend Snippet: Representative image of western blotting of p-p38α (Thr180/Tyr182), p38α, p-IKKα/β (Ser173/180), IKKα and IKKβ in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (A). Representative image of western blotting of p-Rsk1 (Ser380) and Rsk-1 in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice. α-tubulin was used as a loading control (B). Representative image of western blotting of p-Akt (Ser473) and Akt in the liver of young WT and p38α KO mice and in the liver of old WT and p38α KO mice (C). The number of samples per group was 4.

    Techniques Used: Western Blot, Mouse Assay

    19) Product Images from "TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival"

    Article Title: TGF-? Coordinately Activates TAK1/MEK/AKT/NFkB and Smad Pathways to Promote Osteoclast Survival

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2008.06.006

    Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.
    Figure Legend Snippet: Model of proposed signaling pathway. In mature osteoclasts, TGF-β rapidly activates both SMAD and TAK1. TAK1 activation leads to sequential activation of MEK1/2, AKT, NIK, and IKKα/β, leading to phosphorylation of IκB. This causes targeted degradation of IκB, allowing subsequent NFκB nuclear localization to promote osteoclast survival. TGF-β-dependent SMAD2/3 activation and nuclear localization also occurs in parallel. NFκB activates transcription of BCLX L and Mcl-1 whereas SMAD2/3 activate transcription of Mcl-1.

    Techniques Used: Activation Assay

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