anti-iκbα Search Results


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  • 99
    Cell Signaling Technology Inc anti iκbα
    Evidence of miR-222 targeting of Brg1 a , Example of gating that was used to exclude dead cells from flow cytometry analyses in (c), (g), and . b , Example of gating used to distinguish cells with high vs. low levels of <t>IκBα,</t> as analyzed in (c). c , Effect of miRNA overexpression (by viral transduction) on LPS-induced IκBα degradation in iBMDMs, measured by flow cytometry (n=4 independent experiments). d , Sequence and prediction scores of a miR-222 binding site in the Brg1 UTR. e , miR-222 and Brg1 mRNA levels in LPS-stimulated BMDMs (n=3 biologically independent samples). f , Brg1 mRNA levels in resting BMDMs 24 hours after transfection (n=4 biologically independent samples). g , Effect of miRNA overexpression or antagonization (by viral transduction) on BRG1 levels in iBMDMs, observed by flow cytometry. Representative of 4 independent experiments with similar results, quantified in (h). h , Flow cytometry analysis of BRG1 protein levels in transduced iBMDMs (n=4 independent experiments). i , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Brg1 UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (d) (n=3 independent experiments). j , Quantification of average effect of miR-222 mimic transfection on Brg1 -dependent and –independent LPS-response genes (n=3 biologically independent samples). Two-sided Student’s t-test for heteroscedastic values used to compare ratios (miR-222 overexpression/control) at peak LPS-induced expression times for Brg1 -dependent vs. -independent genes. k-l , ChIP for histone H3 acetylation (k), or histone H4 acetylation (l) after LPS stimulation of iBMDMs transduced with overexpression constructs (k-l tested in same n=3 independent experiments). m , Successful deletion of the miR-222 binding site in the Brg1 UTR in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site is highlighted in yellow. n , Effect of miR-222 overexpression (by oligonucleotide transfection) on LPS-induced gene expression in either a RAW cell line in which the Brg1 ) or a cell line in which the binding site was not targeted for deletion (n=5 independent experiments). For all bar graphs, mean +/− SEM is plotted. ** p
    Anti Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti iκbα
    Representative cross sections of the soleus muscle from control ( A ), ligated ( B ), stimulated ( C ), and Lig + Stim ( D ) groups. Muscle fibers fluorescing green in control, ligated, and stimulated sections are expressing EGFP, whereas muscle fibers fluorescing green in Lig + Stim sections are expressing the d.n. <t>IκBα-EGFP</t> fusion protein. E : Muscle-fiber cross-sectional area of ∼250 fibers per muscle (including both transfected and nontransfected fibers) from six muscles per group. * P
    Anti Iκbα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc iκbα
    Quercetin inhibits ZD55-TRAIL induced NF-κB activation in HCC cells. Notes: HuH-7 cells were cultured with ZD55-TRAIL (2MOI), quercetin (10 μM), or ZD55-TRAIL (2MOI) plus quercetin (10 μM) for 48 h. Cell lysates were prepared and ( A ) Western blottingor ( B ) ELISA was performed to examine the changes in <t>IκBα,</t> p65, and p50 expression. GAPDH was used as the loading control. ( C ) Apoptosis-related proteins Bcl-2, FLIP, Bid and Bax were detected by Western blotting after treatment with ZD55-TRAIL (2MOI), quercetin (10 μM), or ZD55-TRAIL (2MOI) plus quercetin (10 μM) for 48 h. Differences in protein levels was determined by densitometry and expressed as percentages. Data are presented as mean ± SD and are representative of three separate experiments. (*Represents P
    Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit anti iκbα
    TRAIL induces the phosphorylation of <t>IκBα.</t> ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). Cells stimulated with macrophage-conditioned medium (MaCM) were used as a positive control. The phosphorylation of IκBα was analyzed by Western blot. α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. ( B ) SGBS adipocytes on day 14 of adipogenic differentiation were treated for 2 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and nuclear extracts were prepared. DNA binding activity of NFκB was analyzed by electrophoretic mobility shift assay (EMSA). One representative experiment out of three performed experiments is presented. ( C ) SGBS adipocytes on day 7 of adipogenic differentiation were transfected with NFκB Firefly luciferase reporter vector and Renilla luciferase control reporter vector. On day 9, cells were treated for 24 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and luciferase activity was determined. Values are means and SEM of 3 different experiments. Unpaired Student´s t-test was used to test for statistical significance. ( D-H ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the IKK inhibitor SC-514 (100 μM). After 6 hours, the phosphorylation of IκBα was analyzed by Western blot ( D ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, the expression of IL-6 ( E ), IL-8 ( F ), MCP-1 ( G ) and CCL-20 ( H ) was assessed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p
    Rabbit Anti Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 239 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti iκbα
    SR reduces DMBA/TPA-induced inflammation in the skin of mice through inactivation of NF-κB in vivo . The skin was removed from mice treated under different conditions. Then, (A) RT-qPCR analysis was used to determine TNF-α, IL-1β, IL-18, IL-6, COX2 and TGF-β1 mRNA levels. (B) Western blot analysis was carried out to evaluate phosphorylated <t>IκBα,</t> IκBα and phosphorylated NF-κB in the skin tissue samples obtained from mice. (C) Representative images of skin after exposure to DMBA/TPA in the absence or presence of SR. The quantification of phosphorylated NF-κB was measured using immunofluorescence analysis. Data are presented as mean ± SEM (n=20). + P
    Anti Iκbα, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology rabbit anti iκbα
    Doxorubicin-induced NF-κB activation enhances DNA damage and apoptosis in A172 glioblastoma cells. (A), Ectopic expression of <t>IκBα-SR.</t> A172 glioblastoma cells were stably transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B), Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of A172 cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C), Inhibition of NF-κB transcriptional activity by IκBα-SR. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα or for 24 hrs with 0.8 μg/ml Doxorubicin and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D), Enhancement of TNFα-induced apoptosis by NF-κB inhibition. A172 cells transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. (E), NF-κB promotes Doxorubicin-induced DNA damage. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (F), NF-κB promotes Doxorubicin-induced apoptosis. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium-iodide stained nuclei. Median (E) or mean (C, D and F) + S.D. of three independent experiments are shown; * P
    Rabbit Anti Iκbα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology rabbit polyclonal anti iκbα
    Doxorubicin-induced NF-κB activation enhances DNA damage and apoptosis in A172 glioblastoma cells. (A), Ectopic expression of <t>IκBα-SR.</t> A172 glioblastoma cells were stably transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B), Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of A172 cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C), Inhibition of NF-κB transcriptional activity by IκBα-SR. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα or for 24 hrs with 0.8 μg/ml Doxorubicin and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D), Enhancement of TNFα-induced apoptosis by NF-κB inhibition. A172 cells transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. (E), NF-κB promotes Doxorubicin-induced DNA damage. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (F), NF-κB promotes Doxorubicin-induced apoptosis. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium-iodide stained nuclei. Median (E) or mean (C, D and F) + S.D. of three independent experiments are shown; * P
    Rabbit Polyclonal Anti Iκbα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti iκb α
    Effect of β-(1→6)-glucan on phosphorylation of <t>IκB-α,</t> ERK, JNK and p38 in murine macrophage cell line J774a.l. (A) Representative results of membranes being probed for phosphorylated protein, re-probed for total protein, stripped and re-probed for loading control. (B) Fold regulation (ratio phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control ( P ≤ 0.05).
    Rabbit Anti Iκb α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti iκb α
    DPQ inhibits degradation of <t>IκB-α</t> and subsequent activation of NF-κB in macrophages. Macrophages were stimulated with LPS for indicated time and lysed for protein extraction. The expressions of IκB-α (A) and phosphorylation of NF-κB p65 (B) were detected by Western blot. The experiment was repeated for 3 times with similar results. Upper panel shows the representative immunoblot and lower panel shows the quantitative analysis of the protein expression. Data are expressed as mean ± SEM; n = 3 * P
    Rabbit Anti Iκb α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti iκbα
    Impaired B cell activation in the HOIP-deficient patient. (A) Flow cytometry analysis of CD80 expression from stimulated PBMCs. PBMCs left unstimulated or stimulated for 3 d as indicated were stained for CD19 and with either an isotype control (red histograms) or an antibody directed against CD80 (blue histograms). CD80 expression values (CD80 median, isotype median) are indicated under the histograms (one experiment). (B) Flow cytometry analysis of CD27/CD38 expression on gated CD19 + cells, upon stimulation of PBMCs for 7 d with CD40L+IL-21 or with medium alone. The gated populations in the figure correspond to CD38 hi and CD27 hi plasmablasts (one experiment). (C) Time course of CD40L-stimulated EBV-immortalized B cells from control, HOIP- (patient), and CD40-deficient (CD40 −/− ) patients, followed by immunoblotting for p-IKK, IKKβ, <t>IκBα,</t> and GAPDH (two independent experiments).
    Anti Iκbα, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit polyclonal anti iκbα
    Impaired B cell activation in the HOIP-deficient patient. (A) Flow cytometry analysis of CD80 expression from stimulated PBMCs. PBMCs left unstimulated or stimulated for 3 d as indicated were stained for CD19 and with either an isotype control (red histograms) or an antibody directed against CD80 (blue histograms). CD80 expression values (CD80 median, isotype median) are indicated under the histograms (one experiment). (B) Flow cytometry analysis of CD27/CD38 expression on gated CD19 + cells, upon stimulation of PBMCs for 7 d with CD40L+IL-21 or with medium alone. The gated populations in the figure correspond to CD38 hi and CD27 hi plasmablasts (one experiment). (C) Time course of CD40L-stimulated EBV-immortalized B cells from control, HOIP- (patient), and CD40-deficient (CD40 −/− ) patients, followed by immunoblotting for p-IKK, IKKβ, <t>IκBα,</t> and GAPDH (two independent experiments).
    Rabbit Polyclonal Anti Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Evidence of miR-222 targeting of Brg1 a , Example of gating that was used to exclude dead cells from flow cytometry analyses in (c), (g), and . b , Example of gating used to distinguish cells with high vs. low levels of IκBα, as analyzed in (c). c , Effect of miRNA overexpression (by viral transduction) on LPS-induced IκBα degradation in iBMDMs, measured by flow cytometry (n=4 independent experiments). d , Sequence and prediction scores of a miR-222 binding site in the Brg1 UTR. e , miR-222 and Brg1 mRNA levels in LPS-stimulated BMDMs (n=3 biologically independent samples). f , Brg1 mRNA levels in resting BMDMs 24 hours after transfection (n=4 biologically independent samples). g , Effect of miRNA overexpression or antagonization (by viral transduction) on BRG1 levels in iBMDMs, observed by flow cytometry. Representative of 4 independent experiments with similar results, quantified in (h). h , Flow cytometry analysis of BRG1 protein levels in transduced iBMDMs (n=4 independent experiments). i , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Brg1 UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (d) (n=3 independent experiments). j , Quantification of average effect of miR-222 mimic transfection on Brg1 -dependent and –independent LPS-response genes (n=3 biologically independent samples). Two-sided Student’s t-test for heteroscedastic values used to compare ratios (miR-222 overexpression/control) at peak LPS-induced expression times for Brg1 -dependent vs. -independent genes. k-l , ChIP for histone H3 acetylation (k), or histone H4 acetylation (l) after LPS stimulation of iBMDMs transduced with overexpression constructs (k-l tested in same n=3 independent experiments). m , Successful deletion of the miR-222 binding site in the Brg1 UTR in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site is highlighted in yellow. n , Effect of miR-222 overexpression (by oligonucleotide transfection) on LPS-induced gene expression in either a RAW cell line in which the Brg1 ) or a cell line in which the binding site was not targeted for deletion (n=5 independent experiments). For all bar graphs, mean +/− SEM is plotted. ** p

    Journal: Nature

    Article Title: Induction of innate immune memory via microRNA targeting of chromatin remodeling factors

    doi: 10.1038/s41586-018-0253-5

    Figure Lengend Snippet: Evidence of miR-222 targeting of Brg1 a , Example of gating that was used to exclude dead cells from flow cytometry analyses in (c), (g), and . b , Example of gating used to distinguish cells with high vs. low levels of IκBα, as analyzed in (c). c , Effect of miRNA overexpression (by viral transduction) on LPS-induced IκBα degradation in iBMDMs, measured by flow cytometry (n=4 independent experiments). d , Sequence and prediction scores of a miR-222 binding site in the Brg1 UTR. e , miR-222 and Brg1 mRNA levels in LPS-stimulated BMDMs (n=3 biologically independent samples). f , Brg1 mRNA levels in resting BMDMs 24 hours after transfection (n=4 biologically independent samples). g , Effect of miRNA overexpression or antagonization (by viral transduction) on BRG1 levels in iBMDMs, observed by flow cytometry. Representative of 4 independent experiments with similar results, quantified in (h). h , Flow cytometry analysis of BRG1 protein levels in transduced iBMDMs (n=4 independent experiments). i , Activity of a luciferase reporter construct in which the luciferase coding sequence is followed by either the complete Brg1 UTR, or a UTR in which the predicted miR-222 binding site has been mutated to the sequence shown in (d) (n=3 independent experiments). j , Quantification of average effect of miR-222 mimic transfection on Brg1 -dependent and –independent LPS-response genes (n=3 biologically independent samples). Two-sided Student’s t-test for heteroscedastic values used to compare ratios (miR-222 overexpression/control) at peak LPS-induced expression times for Brg1 -dependent vs. -independent genes. k-l , ChIP for histone H3 acetylation (k), or histone H4 acetylation (l) after LPS stimulation of iBMDMs transduced with overexpression constructs (k-l tested in same n=3 independent experiments). m , Successful deletion of the miR-222 binding site in the Brg1 UTR in RAW cell clones was confirmed by sequencing genomic DNA of the given cell line. miR-222 binding site is highlighted in yellow. n , Effect of miR-222 overexpression (by oligonucleotide transfection) on LPS-induced gene expression in either a RAW cell line in which the Brg1 ) or a cell line in which the binding site was not targeted for deletion (n=5 independent experiments). For all bar graphs, mean +/− SEM is plotted. ** p

    Article Snippet: Either anti-IκBα (L35A5, Cell Signaling 4814), anti-Brg1 (H88, Santa Cruz sc-10768), or Rabbit mAb IgG Isotype Control (Cell Signaling 3900) was added, and cells were incubated for an additional 20 minutes at room temperature.

    Techniques: Flow Cytometry, Cytometry, Over Expression, Transduction, Sequencing, Binding Assay, Transfection, Activity Assay, Luciferase, Construct, Expressing, Chromatin Immunoprecipitation, Clone Assay

    Expressions of epithelial NF-κB and inflammatory parameters in IR-ALI. (A) IκB-α and nuclear phosphorylated NF-κB p65 levels in MLE-12 cells treated with BMT. (B) Hematoxylin and eosin staining for lung tissue (200× magnification). (C) Lung injury score in IR model. (D) TNF-α level in the perfusate in IR model. TATA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD ( n = 5 per group). * P

    Journal: Frontiers in Immunology

    Article Title: Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways

    doi: 10.3389/fimmu.2018.02049

    Figure Lengend Snippet: Expressions of epithelial NF-κB and inflammatory parameters in IR-ALI. (A) IκB-α and nuclear phosphorylated NF-κB p65 levels in MLE-12 cells treated with BMT. (B) Hematoxylin and eosin staining for lung tissue (200× magnification). (C) Lung injury score in IR model. (D) TNF-α level in the perfusate in IR model. TATA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD ( n = 5 per group). * P

    Article Snippet: The blots were incubated overnight with primary antibodies anti-α-ENaC, anti-TRAF6 (1:200, Santa Cruz Biotechnology, USA), anti-phosphorylated-p38, anti-p38, anti-phosphorylated-Erk, anti-Erk, anti-phosphorylated-JNK, anti-JNK, anti-phosphorylated-NF-κB p65, anti-IκB-α, anti-TATA (1:1,000, Cell Signaling Technology, USA), and anti-β-actin (1:10,000, Sigma Chemical Company, USA).

    Techniques: Staining

    Evo suppresses NF-κB/MAPK pathways. Evo suppressed the expression of NF-κB and MAPK signaling pathways in mice with staphylococcal pneumonia following cytomegalovirus reactivation. (A) Expression levels of p-IκBα, IκBα, and nuclear and cytosolic p65NF-κB; and the (B) phosphorylation of ERK, JNK and p38 were detected by western blot analysis. The graph shows the quantification of results normalized to β-actin or Lamin A levels. Data are shown as the mean ± standard deviation. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: Evodiamine alleviates severe pneumonia induced by methicillin-susceptible Staphylococcus aureus following cytomegalovirus reactivation through suppressing NF-κB and MAPKs

    doi: 10.3892/ijmm.2018.3929

    Figure Lengend Snippet: Evo suppresses NF-κB/MAPK pathways. Evo suppressed the expression of NF-κB and MAPK signaling pathways in mice with staphylococcal pneumonia following cytomegalovirus reactivation. (A) Expression levels of p-IκBα, IκBα, and nuclear and cytosolic p65NF-κB; and the (B) phosphorylation of ERK, JNK and p38 were detected by western blot analysis. The graph shows the quantification of results normalized to β-actin or Lamin A levels. Data are shown as the mean ± standard deviation. ## P

    Article Snippet: Antibodies against IκBα (cat. no. 9242S; 1:1,000; Cell Signaling Technology, Inc.), p-IκBα (cat. no. 8219; 1:1,000; Cell Signaling Technology, Inc.), NF-κB p65 (cat. no. ab16502; 1:500; Abcam), JNK (cat. no. ab176662; 1:1,000; Abcam), p-JNK (cat. no. ab207477; 1:1,000; Abcam), Erk (cat. no. ab176641; 1:1,000; Abcam), p-Erk (cat. no. ab192591; 1:1,000; Abcam), p38 (cat. no. ab32142; 1:1,000; Abcam), p-p38 (cat. no. ab178867; 1:1,000; Abcam), β-actin (cat. no. ab8226; 1:3,000; Abcam) and Lamin A (cat. no. ab26300; 1:3,000; Abcam) were used. β-actin and Lamin A were used as internal controls.

    Techniques: Expressing, Mouse Assay, Western Blot, Standard Deviation

    Nuclear accumulation of p65 is suppressed in Sirt7 -deficient cells. ( A ) Protein expression of p65, phospho-p65, and IκBα in whole cell lysates of shRNA-introduced NRK-52E cells with or without 1-h cisplatin (30 μM) treatment was evaluated by western blot analysis. β-actin was used as a loading control. ( B . ( C ) Intracellular localization of p65. NRK-52E cells with or without 1-h cisplatin (30 μM) treatment were transfected with the pCI-HA-p65 plasmid, and p65 expression was evaluated by HA immunostaining. For nuclear staining, Hoechst 33342 was used. Relative fluorescence intensity is represented by the ratio of nuclear fluorescence intensity in the ROI to cytoplasmic fluorescence intensity in the ROI as measured by ImageJ software (n = 50/group). *p

    Journal: Scientific Reports

    Article Title: Sirtuin 7 Deficiency Ameliorates Cisplatin-induced Acute Kidney Injury Through Regulation of the Inflammatory Response

    doi: 10.1038/s41598-018-24257-7

    Figure Lengend Snippet: Nuclear accumulation of p65 is suppressed in Sirt7 -deficient cells. ( A ) Protein expression of p65, phospho-p65, and IκBα in whole cell lysates of shRNA-introduced NRK-52E cells with or without 1-h cisplatin (30 μM) treatment was evaluated by western blot analysis. β-actin was used as a loading control. ( B . ( C ) Intracellular localization of p65. NRK-52E cells with or without 1-h cisplatin (30 μM) treatment were transfected with the pCI-HA-p65 plasmid, and p65 expression was evaluated by HA immunostaining. For nuclear staining, Hoechst 33342 was used. Relative fluorescence intensity is represented by the ratio of nuclear fluorescence intensity in the ROI to cytoplasmic fluorescence intensity in the ROI as measured by ImageJ software (n = 50/group). *p

    Article Snippet: Primary antibodies included an anti-SIRT7 antibody (#5360, Cell Signaling Technology), anti-cleaved caspase 3 antibody (#9661, Cell Signaling Technology), anti-p65 antibody (#4764, Cell Signaling Technology), anti-phospho-p65 antibody (#3031, Cell Signaling Technology), anti-IκBα antibody (#9242, Cell Signaling Technology), anti-β-actin antibody (A5060, Sigma Aldrich), anti-GAPDH antibody (#2118, Cell Signaling Technology), and anti-histone H3 antibody (#39163, Active Motif).

    Techniques: Expressing, shRNA, Western Blot, Transfection, Plasmid Preparation, Immunostaining, Staining, Fluorescence, Software

    Patulin inhibited TNF-α-induced p65 nuclear translocation without acting on IκBα phosphorylation or degradation. ( a ) NF-κB p65 immunocytochemical translocation pattern in A549 cells following 30 min incubation with either DMSO, 20 ng/mL TNF-α or TNF-α + 1.5 μM patulin (Pat). DAPI counterstaining (0.1 μg/mL) was used to confirm nuclear localization. Quantification of relative p65 nuclear translocation was performed from the immunocytochemistry pictures ( n = 3). * p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Lung Cancer Chemopreventive Activity of Patulin Isolated from Penicillium vulpinum

    doi: 10.3390/molecules23030636

    Figure Lengend Snippet: Patulin inhibited TNF-α-induced p65 nuclear translocation without acting on IκBα phosphorylation or degradation. ( a ) NF-κB p65 immunocytochemical translocation pattern in A549 cells following 30 min incubation with either DMSO, 20 ng/mL TNF-α or TNF-α + 1.5 μM patulin (Pat). DAPI counterstaining (0.1 μg/mL) was used to confirm nuclear localization. Quantification of relative p65 nuclear translocation was performed from the immunocytochemistry pictures ( n = 3). * p

    Article Snippet: After protein denaturation, 20 µg of proteins were loaded on 12.5% bis-acrylamide gels, and electrophoresis was performed at 100 V. Proteins were then transferred to a PVDF membrane for 40 min at 10 V. After the transfer, membranes were blocked in 5% non-fat milk for 60 min under agitation, and incubated overnight with the primary antibodies (1:1000 in 1% non-fat milk), either mouse anti-IκBα or rabbit anti-phospho-IκBα (Cell Signaling Technology) at 4 °C.

    Techniques: Translocation Assay, Incubation, Immunocytochemistry

    CM from L. plantarum , L. acidophilus , and B. infantis suppressed NF-κB signaling and preserved IκBα expression. Intestines from healthy dam-fed controls and experimental intestinal injury pups treated with vehicle or different CM were IHC stained for the active/phosphorylated form of NF-κB p65 (p-NF-κB) and IκBα or lysed for immunoblotting for IκBα. A. Representative IHC staining (at 400×) shows that CM decreased IEC NF-κB activity/nuclear p-NF-κB (arrows) in experimental pups with or without intestinal injury (n = 3 per group). B. NF-κB activity from A. was quantified by percentage of nuclear p-NF-κB-positive IEC in three fields of each intestinal section (n = 3). C. Representative IHC staining shows La/Bi CM and all CM protection of IEC IκBα expression in experimental pups with or without intestinal injury, respectively, (n = 3 per group). D. IHC intensity of IκBα from C. was scored on a 0–4 scale to relatively quantify levels of IκBα. E. Immunoblotting shows CM protection of IEC IκBα expression in experimental pups. Intestines of vehicle- and CM-treated experimental pups with disease and healthy dam-fed pups were lysed and subjected to IκBα immunoblotting. IκBα protein levels were quantified by densitometry using ImageJ software and normalized to GAPDH and presented in F. Results are presented as mean of percentages, scores, or relative density ± SE. “*” depicts p

    Journal: PLoS ONE

    Article Title: Synergistic Protection of Combined Probiotic Conditioned Media against Neonatal Necrotizing Enterocolitis-Like Intestinal Injury

    doi: 10.1371/journal.pone.0065108

    Figure Lengend Snippet: CM from L. plantarum , L. acidophilus , and B. infantis suppressed NF-κB signaling and preserved IκBα expression. Intestines from healthy dam-fed controls and experimental intestinal injury pups treated with vehicle or different CM were IHC stained for the active/phosphorylated form of NF-κB p65 (p-NF-κB) and IκBα or lysed for immunoblotting for IκBα. A. Representative IHC staining (at 400×) shows that CM decreased IEC NF-κB activity/nuclear p-NF-κB (arrows) in experimental pups with or without intestinal injury (n = 3 per group). B. NF-κB activity from A. was quantified by percentage of nuclear p-NF-κB-positive IEC in three fields of each intestinal section (n = 3). C. Representative IHC staining shows La/Bi CM and all CM protection of IEC IκBα expression in experimental pups with or without intestinal injury, respectively, (n = 3 per group). D. IHC intensity of IκBα from C. was scored on a 0–4 scale to relatively quantify levels of IκBα. E. Immunoblotting shows CM protection of IEC IκBα expression in experimental pups. Intestines of vehicle- and CM-treated experimental pups with disease and healthy dam-fed pups were lysed and subjected to IκBα immunoblotting. IκBα protein levels were quantified by densitometry using ImageJ software and normalized to GAPDH and presented in F. Results are presented as mean of percentages, scores, or relative density ± SE. “*” depicts p

    Article Snippet: The specimens were blocked with 5% BSA in TBST (Tris-buffered saline with 0.05% vol/vol Tween-20) followed by incubation at 4°C overnight with p-NF-κB, IκBα antibodies (Cell Signaling), or control IgGs isolated before immunization from the same animal species used to generate the p-NF-κB or IκBα antibodies.

    Techniques: Expressing, Immunohistochemistry, Staining, Activity Assay, Software

    Functional analyses of BCL10-BCL10 interfaces in Jurkat T-cells. a , b BCL10 KO Jurkat T-cells lentivirally reconstituted with BCL10 wt, R42E, R36E or R42A constructs were stimulated P/I for 20 min and CBM complex formation was monitored by BCL10-IP ( a ) and Strep-tag II (ST) pull down (PD) ( b ). c , d BCL10 KO Jurkat T-cells reconstituted with BCL10 wt or BCL10-BCL10 interface mutants were stimulated with P/I for the times indicated. NF-κB activation was determined by IκBα WB and EMSA. Activation of the MALT1 protease was monitored by WB analyses of the substrate cleavage. Asterisks indicate non-specific cross-reactivity of the BCL10 antibody

    Journal: Nature Communications

    Article Title: Molecular architecture and regulation of BCL10-MALT1 filaments

    doi: 10.1038/s41467-018-06573-8

    Figure Lengend Snippet: Functional analyses of BCL10-BCL10 interfaces in Jurkat T-cells. a , b BCL10 KO Jurkat T-cells lentivirally reconstituted with BCL10 wt, R42E, R36E or R42A constructs were stimulated P/I for 20 min and CBM complex formation was monitored by BCL10-IP ( a ) and Strep-tag II (ST) pull down (PD) ( b ). c , d BCL10 KO Jurkat T-cells reconstituted with BCL10 wt or BCL10-BCL10 interface mutants were stimulated with P/I for the times indicated. NF-κB activation was determined by IκBα WB and EMSA. Activation of the MALT1 protease was monitored by WB analyses of the substrate cleavage. Asterisks indicate non-specific cross-reactivity of the BCL10 antibody

    Article Snippet: After permeabilization (0.1% saponine), cells were stained using mouse anti-IκBα antibody (L35A5, CST) and anti-mouse IgG1 FITC (BD).

    Techniques: Functional Assay, Construct, Strep-tag, Activation Assay, Western Blot

    Representative cross sections of the soleus muscle from control ( A ), ligated ( B ), stimulated ( C ), and Lig + Stim ( D ) groups. Muscle fibers fluorescing green in control, ligated, and stimulated sections are expressing EGFP, whereas muscle fibers fluorescing green in Lig + Stim sections are expressing the d.n. IκBα-EGFP fusion protein. E : Muscle-fiber cross-sectional area of ∼250 fibers per muscle (including both transfected and nontransfected fibers) from six muscles per group. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: IκBα degradation is necessary for skeletal muscle atrophy associated with contractile claudication

    doi: 10.1152/ajpregu.00728.2010

    Figure Lengend Snippet: Representative cross sections of the soleus muscle from control ( A ), ligated ( B ), stimulated ( C ), and Lig + Stim ( D ) groups. Muscle fibers fluorescing green in control, ligated, and stimulated sections are expressing EGFP, whereas muscle fibers fluorescing green in Lig + Stim sections are expressing the d.n. IκBα-EGFP fusion protein. E : Muscle-fiber cross-sectional area of ∼250 fibers per muscle (including both transfected and nontransfected fibers) from six muscles per group. * P

    Article Snippet: The following primary antibodies were used: anti-GFP (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA); anti-IκBα, 1:1,000 (sc-371; Santa Cruz Biotechnology); anti-MuRF1, 1:500 (MP3401; ECM Biosciences, Versailles, KY); anti-atrogin-1, 1:500 (AP2041; ECM Biosciences); anti-tubulin, 1:1,000 (T6074; Sigma-Aldrich, St. Louis, MO).

    Techniques: Expressing, Transfection

    Representative Western blot with one sample from each group and bar graph of atrogin-1 ( A ) and MuRF1 ( B ) protein expression following repeated bouts of contractile claudication. Both atrogin-1 and MuRF1 protein levels are significantly increased in the Lig + Stim group, and this increase is abolished in Lig + Stim muscles expressing the d.n. IκBα. Bars represent means ± SE of six muscles per group. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: IκBα degradation is necessary for skeletal muscle atrophy associated with contractile claudication

    doi: 10.1152/ajpregu.00728.2010

    Figure Lengend Snippet: Representative Western blot with one sample from each group and bar graph of atrogin-1 ( A ) and MuRF1 ( B ) protein expression following repeated bouts of contractile claudication. Both atrogin-1 and MuRF1 protein levels are significantly increased in the Lig + Stim group, and this increase is abolished in Lig + Stim muscles expressing the d.n. IκBα. Bars represent means ± SE of six muscles per group. * P

    Article Snippet: The following primary antibodies were used: anti-GFP (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA); anti-IκBα, 1:1,000 (sc-371; Santa Cruz Biotechnology); anti-MuRF1, 1:500 (MP3401; ECM Biosciences, Versailles, KY); anti-atrogin-1, 1:500 (AP2041; ECM Biosciences); anti-tubulin, 1:1,000 (T6074; Sigma-Aldrich, St. Louis, MO).

    Techniques: Western Blot, Expressing

    mRNA levels of atrogin-1 ( A ), MuRF1 ( B ), Nedd4 ( C ), and cathepsin L ( D ) following a single bout of contractile claudication. MuRF1, cathepsin L, and Nedd4 mRNA expression is significantly increased in the Lig + Stim group, and this increase is abolished in Lig + Stim muscles expressing the d.n. IκBα. Atrogin-1 mRNA expression is unchanged across all groups. Bars represent means ± SE of six muscles per group. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: IκBα degradation is necessary for skeletal muscle atrophy associated with contractile claudication

    doi: 10.1152/ajpregu.00728.2010

    Figure Lengend Snippet: mRNA levels of atrogin-1 ( A ), MuRF1 ( B ), Nedd4 ( C ), and cathepsin L ( D ) following a single bout of contractile claudication. MuRF1, cathepsin L, and Nedd4 mRNA expression is significantly increased in the Lig + Stim group, and this increase is abolished in Lig + Stim muscles expressing the d.n. IκBα. Atrogin-1 mRNA expression is unchanged across all groups. Bars represent means ± SE of six muscles per group. * P

    Article Snippet: The following primary antibodies were used: anti-GFP (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA); anti-IκBα, 1:1,000 (sc-371; Santa Cruz Biotechnology); anti-MuRF1, 1:500 (MP3401; ECM Biosciences, Versailles, KY); anti-atrogin-1, 1:500 (AP2041; ECM Biosciences); anti-tubulin, 1:1,000 (T6074; Sigma-Aldrich, St. Louis, MO).

    Techniques: Expressing

    NF-κB transcriptional activity following a single bout of contractile claudication. A : Preliminary data (bars represent means ± SE of 3 muscles per group) to determine the time course of NF-κB transactivation following a single bout of contractile claudication. B : NF-κB activity is significantly increased in the soleus muscle of Lig + Stim animals 1 h following the stimulation. Expression of the dominant-negative (d.n.) IκBα in Lig + Stim muscles abolished NF-κB activity. C : Endogenous IκBα protein expression and ectopically expressed d.n. IκBα protein expression, showing a 30% decrease in endogenous IκBα protein levels in the Lig + Stim group. Data for Lig + Stim (d.n. IκBα) are presented as a stacked bar to show endogenous IκBα and d.n. IκBα-EGFP expression. Bars for B and C represent means ± SE of six muscles per group. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: IκBα degradation is necessary for skeletal muscle atrophy associated with contractile claudication

    doi: 10.1152/ajpregu.00728.2010

    Figure Lengend Snippet: NF-κB transcriptional activity following a single bout of contractile claudication. A : Preliminary data (bars represent means ± SE of 3 muscles per group) to determine the time course of NF-κB transactivation following a single bout of contractile claudication. B : NF-κB activity is significantly increased in the soleus muscle of Lig + Stim animals 1 h following the stimulation. Expression of the dominant-negative (d.n.) IκBα in Lig + Stim muscles abolished NF-κB activity. C : Endogenous IκBα protein expression and ectopically expressed d.n. IκBα protein expression, showing a 30% decrease in endogenous IκBα protein levels in the Lig + Stim group. Data for Lig + Stim (d.n. IκBα) are presented as a stacked bar to show endogenous IκBα and d.n. IκBα-EGFP expression. Bars for B and C represent means ± SE of six muscles per group. * P

    Article Snippet: The following primary antibodies were used: anti-GFP (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA); anti-IκBα, 1:1,000 (sc-371; Santa Cruz Biotechnology); anti-MuRF1, 1:500 (MP3401; ECM Biosciences, Versailles, KY); anti-atrogin-1, 1:500 (AP2041; ECM Biosciences); anti-tubulin, 1:1,000 (T6074; Sigma-Aldrich, St. Louis, MO).

    Techniques: Activity Assay, Expressing, Dominant Negative Mutation

    NF-κB transcriptional activity following repeated bouts of contractile claudication. A : NF-κB activity is significantly increased in the stim group and in the Lig + Stim group, compared with all groups. The increase in NF-κB activity in the Lig + Stim muscles is abolished in Lig + Stim muscles expressing the d.n. IκBα. B : representative Western blot with one sample from each group and bar graph of mean changes from six muscles per group of endogenous IκBα protein expression (37 kDa) and ectopically expressed d.n. IκBα-EGFP protein expression (64 kDa). Endogenous IκBα protein expression is down-regulated in the Lig + Stim group. Data for Lig + Stim (d.n. IκBα) are presented as a stacked bar to show endogenous IκBα and d.n. IκBα-EGFP expression. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: IκBα degradation is necessary for skeletal muscle atrophy associated with contractile claudication

    doi: 10.1152/ajpregu.00728.2010

    Figure Lengend Snippet: NF-κB transcriptional activity following repeated bouts of contractile claudication. A : NF-κB activity is significantly increased in the stim group and in the Lig + Stim group, compared with all groups. The increase in NF-κB activity in the Lig + Stim muscles is abolished in Lig + Stim muscles expressing the d.n. IκBα. B : representative Western blot with one sample from each group and bar graph of mean changes from six muscles per group of endogenous IκBα protein expression (37 kDa) and ectopically expressed d.n. IκBα-EGFP protein expression (64 kDa). Endogenous IκBα protein expression is down-regulated in the Lig + Stim group. Data for Lig + Stim (d.n. IκBα) are presented as a stacked bar to show endogenous IκBα and d.n. IκBα-EGFP expression. * P

    Article Snippet: The following primary antibodies were used: anti-GFP (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA); anti-IκBα, 1:1,000 (sc-371; Santa Cruz Biotechnology); anti-MuRF1, 1:500 (MP3401; ECM Biosciences, Versailles, KY); anti-atrogin-1, 1:500 (AP2041; ECM Biosciences); anti-tubulin, 1:1,000 (T6074; Sigma-Aldrich, St. Louis, MO).

    Techniques: Activity Assay, Expressing, Western Blot

    mRNA levels of atrogin-1 ( A ), MuRF1 ( B ), Nedd4 ( C ), and cathepsin L ( D ) following repeated bouts of contractile claudication. Each gene is significantly increased in the Lig + Stim group, and this increase is abolished in Lig + Stim muscles expressing the d.n. IκBα. Bars represent means ± SE of six muscles per group. * P

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: IκBα degradation is necessary for skeletal muscle atrophy associated with contractile claudication

    doi: 10.1152/ajpregu.00728.2010

    Figure Lengend Snippet: mRNA levels of atrogin-1 ( A ), MuRF1 ( B ), Nedd4 ( C ), and cathepsin L ( D ) following repeated bouts of contractile claudication. Each gene is significantly increased in the Lig + Stim group, and this increase is abolished in Lig + Stim muscles expressing the d.n. IκBα. Bars represent means ± SE of six muscles per group. * P

    Article Snippet: The following primary antibodies were used: anti-GFP (sc-8334; Santa Cruz Biotechnology, Santa Cruz, CA); anti-IκBα, 1:1,000 (sc-371; Santa Cruz Biotechnology); anti-MuRF1, 1:500 (MP3401; ECM Biosciences, Versailles, KY); anti-atrogin-1, 1:500 (AP2041; ECM Biosciences); anti-tubulin, 1:1,000 (T6074; Sigma-Aldrich, St. Louis, MO).

    Techniques: Expressing

    Subcellular localization of DNA-PKcs, IκBα, and the RelA subunit of NF-κB. HeLa (A) 50D (B), and 100E (C) cells were stained by immunofluorescence with a monoclonal antibody directed against DNA-PKcs (25-4) (left panel), a polyclonal antibody directed against IκBα (C-21) (middle panel), or a polyclonal antibody directed against RelA (C-20) (right panel), followed by goat anti-mouse FITC or goat anti-rabbit FITC conjugate. Preparations were analyzed by confocal microscopy.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Subcellular localization of DNA-PKcs, IκBα, and the RelA subunit of NF-κB. HeLa (A) 50D (B), and 100E (C) cells were stained by immunofluorescence with a monoclonal antibody directed against DNA-PKcs (25-4) (left panel), a polyclonal antibody directed against IκBα (C-21) (middle panel), or a polyclonal antibody directed against RelA (C-20) (right panel), followed by goat anti-mouse FITC or goat anti-rabbit FITC conjugate. Preparations were analyzed by confocal microscopy.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Staining, Immunofluorescence, Confocal Microscopy

    NF-κB DNA-binding activity in SCID and DNA-PKcs-complemented cells. (A) Nuclear extracts were prepared from CB-17 (parental), SCID-St (SCID), 100E (DNA-PKcs-complemented SCID cells), and 50D (non-DNA-PKcs-complemented SCID cells) cells and subjected to gel retardation analysis using NF-κB oligonucleotides. Results for probe alone (lane 1) and nuclear extract prepared from CB-17 (lane 2), SCID-St (lane 3), 100E (lane 4), and 50D (lane 5) cells are shown. Nuclear extract prepared from SCID-St cells (lane 6) was analyzed in these gel retardation assays in the presence of preimmune serum (lane 7), antibody directed against RelA (lane 8), or the NF-κB p50 subunit (lane 9). (B) Gel retardation analysis with an SP1 probe either alone (lane 1) or in the presence of nuclear extract prepared from CB-17 (lane 2), SCID-St (lane 3), 100E (lane 4), or 50D (lane 5) cells was performed. (C) The recombinant NF-κB subunits p50 and p65 (RelA) were purified following baculovirus expression and analyzed by gel retardation with NF-κB binding site oligonucleotides. Probe alone (lane 1) and the p50-p65 proteins without the addition of IκBα (lane 2) are shown, as are p50-p65 with 50, 100, or 200 ng of either purified IκBα (lanes 3 to 5, respectively) or DNA-PK-phosphorylated IκBα (lanes 6 to 8, respectively), which was added to the p50-p65 proteins approximately 30 min prior to the addition of the NF-κB probe. The results for p50-p65 proteins in the presence of antibody directed against p65 (RelA) (lane 9) or CBP (lane 10) are also indicated. (D) Western blot analysis was performed with cytoplasmic (odd-numbered lanes) or nuclear (even-numbered lanes) extracts prepared from CB-17 (lanes 1 and 2), SCID-St (lanes 3 and 4), 100E (lanes 5 and 6), and 50D (lanes 7 and 8) cells with a monoclonal antibody directed against DNA-PKcs (42-26). (E) Western blot analysis was performed on cytoplasmic extracts prepared from CB-17, SCID-St, 100E, and 50D cells with polyclonal antibody directed against IκBα.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: NF-κB DNA-binding activity in SCID and DNA-PKcs-complemented cells. (A) Nuclear extracts were prepared from CB-17 (parental), SCID-St (SCID), 100E (DNA-PKcs-complemented SCID cells), and 50D (non-DNA-PKcs-complemented SCID cells) cells and subjected to gel retardation analysis using NF-κB oligonucleotides. Results for probe alone (lane 1) and nuclear extract prepared from CB-17 (lane 2), SCID-St (lane 3), 100E (lane 4), and 50D (lane 5) cells are shown. Nuclear extract prepared from SCID-St cells (lane 6) was analyzed in these gel retardation assays in the presence of preimmune serum (lane 7), antibody directed against RelA (lane 8), or the NF-κB p50 subunit (lane 9). (B) Gel retardation analysis with an SP1 probe either alone (lane 1) or in the presence of nuclear extract prepared from CB-17 (lane 2), SCID-St (lane 3), 100E (lane 4), or 50D (lane 5) cells was performed. (C) The recombinant NF-κB subunits p50 and p65 (RelA) were purified following baculovirus expression and analyzed by gel retardation with NF-κB binding site oligonucleotides. Probe alone (lane 1) and the p50-p65 proteins without the addition of IκBα (lane 2) are shown, as are p50-p65 with 50, 100, or 200 ng of either purified IκBα (lanes 3 to 5, respectively) or DNA-PK-phosphorylated IκBα (lanes 6 to 8, respectively), which was added to the p50-p65 proteins approximately 30 min prior to the addition of the NF-κB probe. The results for p50-p65 proteins in the presence of antibody directed against p65 (RelA) (lane 9) or CBP (lane 10) are also indicated. (D) Western blot analysis was performed with cytoplasmic (odd-numbered lanes) or nuclear (even-numbered lanes) extracts prepared from CB-17 (lanes 1 and 2), SCID-St (lanes 3 and 4), 100E (lanes 5 and 6), and 50D (lanes 7 and 8) cells with a monoclonal antibody directed against DNA-PKcs (42-26). (E) Western blot analysis was performed on cytoplasmic extracts prepared from CB-17, SCID-St, 100E, and 50D cells with polyclonal antibody directed against IκBα.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Recombinant, Purification, Expressing, Western Blot

    IκBα and NF-κB regulation in SCID cells. Cytoplasmic extracts were prepared from either the SCID cell line SF19 (lanes 1 to 5) or NIH 3T3 cells (lanes 6 to 10) that were untreated (lanes 1 and 6) or treated with TPA and ionomycin for 15 (lanes 2 and 7), 30 (lanes 3 and 8), 60 (lanes 4 and 9), or 90 (lanes 5 and 10) min. Western blot analysis was performed with 20 μg of each of these extracts by using rabbit polyclonal antibody directed against either IκBα (A) or the p89 subunit of TFIIH (B). Nuclear extracts were prepared from NIH 3T3 cells (lane 2), SF19 cells (lane 3), the wild-type equine cell line CRL6288 (lane 4), and the equine SCID cell line 1863 (lane 5) and subjected to gel retardation analysis with either an oligonucleotide containing an NF-κB (C) or an SP1 (D) binding site, followed by autoradiography. An analysis of probe alone is also shown (lane 1). For panel C, nuclear extract was prepared from Jurkat cells that were not treated (lane 6) or treated with TPA and ionomycin for 10 (lane 7) or 30 (lane 8) min. Antibody to the RelA (p65) (lane 10) or p50 (lane 10) subunits of NF-κB or CBP (lane 11) was added to the gel retardation assay mixtures for supershift analysis.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: IκBα and NF-κB regulation in SCID cells. Cytoplasmic extracts were prepared from either the SCID cell line SF19 (lanes 1 to 5) or NIH 3T3 cells (lanes 6 to 10) that were untreated (lanes 1 and 6) or treated with TPA and ionomycin for 15 (lanes 2 and 7), 30 (lanes 3 and 8), 60 (lanes 4 and 9), or 90 (lanes 5 and 10) min. Western blot analysis was performed with 20 μg of each of these extracts by using rabbit polyclonal antibody directed against either IκBα (A) or the p89 subunit of TFIIH (B). Nuclear extracts were prepared from NIH 3T3 cells (lane 2), SF19 cells (lane 3), the wild-type equine cell line CRL6288 (lane 4), and the equine SCID cell line 1863 (lane 5) and subjected to gel retardation analysis with either an oligonucleotide containing an NF-κB (C) or an SP1 (D) binding site, followed by autoradiography. An analysis of probe alone is also shown (lane 1). For panel C, nuclear extract was prepared from Jurkat cells that were not treated (lane 6) or treated with TPA and ionomycin for 10 (lane 7) or 30 (lane 8) min. Antibody to the RelA (p65) (lane 10) or p50 (lane 10) subunits of NF-κB or CBP (lane 11) was added to the gel retardation assay mixtures for supershift analysis.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Western Blot, Electrophoretic Mobility Shift Assay, Binding Assay, Autoradiography

    Overexpression or decreased degradation of IκBα reduces NF-κB DNA-binding activity in SCID cells. A wild-type IκBα cDNA or an IκBα cDNA with mutations of serine residues 32 and 36 to alanine were cloned into adenovirus. These viruses and a control adenovirus containing the β-galactosidase gene were used to infect NIH 3T3 and SF19 cells at a multiplicity of infection of 10 and harvested 24 h later. Nuclear extracts were prepared from mock- and adenovirus-infected cells and subjected to gel retardation analysis with oligonucleotides containing NF-κB (A) or SP1 (B) binding sites. Gel retardation analysis was performed with nuclear extracts isolated from uninfected cells (lanes 1 and 5), cells infected with adenovirus containing the β-galactosidase gene (lanes 2 and 6), cells infected with adenovirus containing wild-type IκBα (lanes 3 and 7), and cells infected with adenovirus containing IκBα with serine residues 32 and 36 mutated to alanine (lanes 4 and 8). (C) NIH 3T3 (lanes 1 and 2) and SF19 (lanes 3 and 4) cells were treated with 50 μM TPCK (Sigma) for 45 min. Nuclear extracts were prepared from nontreated cells (lanes 1 and 3) and TPCK-treated cells (lanes 2 and 4) and subjected to gel retardation analysis with a double-stranded NF-κB binding oligonucleotide.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Overexpression or decreased degradation of IκBα reduces NF-κB DNA-binding activity in SCID cells. A wild-type IκBα cDNA or an IκBα cDNA with mutations of serine residues 32 and 36 to alanine were cloned into adenovirus. These viruses and a control adenovirus containing the β-galactosidase gene were used to infect NIH 3T3 and SF19 cells at a multiplicity of infection of 10 and harvested 24 h later. Nuclear extracts were prepared from mock- and adenovirus-infected cells and subjected to gel retardation analysis with oligonucleotides containing NF-κB (A) or SP1 (B) binding sites. Gel retardation analysis was performed with nuclear extracts isolated from uninfected cells (lanes 1 and 5), cells infected with adenovirus containing the β-galactosidase gene (lanes 2 and 6), cells infected with adenovirus containing wild-type IκBα (lanes 3 and 7), and cells infected with adenovirus containing IκBα with serine residues 32 and 36 mutated to alanine (lanes 4 and 8). (C) NIH 3T3 (lanes 1 and 2) and SF19 (lanes 3 and 4) cells were treated with 50 μM TPCK (Sigma) for 45 min. Nuclear extracts were prepared from nontreated cells (lanes 1 and 3) and TPCK-treated cells (lanes 2 and 4) and subjected to gel retardation analysis with a double-stranded NF-κB binding oligonucleotide.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Over Expression, Binding Assay, Activity Assay, Clone Assay, Infection, Electrophoretic Mobility Shift Assay, Isolation

    Specificity of DNA-PK phosphorylation of IκBα and IκBβ. (A) Kinase assays were performed with purified DNA-PK and substrates including α-casein (lanes 1 and 2), GST (lanes 3 and 4), or GST fusion proteins containing wild-type IκBα (lanes 5 and 6), this construct with a mutation of serine residues 32 and 36 to alanine (lanes 7 and 8), IκBα with amino acids 1 to 53 deleted (lanes 9 and 10), or IκBα with amino acids 139 to 317 deleted (lanes 11 and 12). The kinase assays were performed in the absence (−) or presence (+) of sheared salmon sperm DNA (0.5 μg), as indicated. (B) GST fusion proteins containing either wild-type IκBα (lanes 1 and 2), IκBα (amino acids [aa] 1 to 275) with the majority of the PEST domain deleted (lanes 3 and 4), full-length IκBα with a mutation of threonine residue 291 to alanine (lanes 5 and 6), IκBα with mutations of serine residue 283 and threonine residues 291 and 299 to alanine (lanes 7 and 8), or IκBα with threonine residues 273, 291, and 299 and serine residue 283 changed to alanine (lanes 9 and 10) were analyzed in kinase assays in either the absence (−) or presence (+) of double-stranded DNA and purified DNA-PK. (C) GST fusion proteins containing either wild-type IκBα (lane 1), full-length IκBα with serine residues 32 and 36 changed to alanine (lane 2), IκBα (aa 1 to 138) with a deletion of aa 139 to 317 (lane 3), this same construct with serine residues 32 and 36 mutated to alanine (lane 4), IκBα (aa 1 to 138) with serine residue 32 changed to alanine (lane 5), IκBα (aa 1 to 138) with serine residue 36 changed to alanine (lane 6), IκBα (aa 1 to 138) with tyrosine residue 42 changed to phenylalanine (lane 7), and IκBα (aa 1 to 138) with mutations of serine residues 32 and 36 to alanine and tyrosine 42 to phenylalanine (lane 8) were analyzed in kinase assays in the presence of double-stranded DNA and purified DNA-PK. (D) GST fusion proteins containing wild-type IκBβ (lane 1), this same construct with serine residues 19 and 23 mutated to alanine (lane 2), a truncated IκBβ construct (aa 1 to 305) (lane 3), or a truncated IκBβ construct with serine residues 19 and 23 changed to alanine (lane 4) were also analyzed in kinase assays with double-stranded DNA and purified DNA-PK.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Specificity of DNA-PK phosphorylation of IκBα and IκBβ. (A) Kinase assays were performed with purified DNA-PK and substrates including α-casein (lanes 1 and 2), GST (lanes 3 and 4), or GST fusion proteins containing wild-type IκBα (lanes 5 and 6), this construct with a mutation of serine residues 32 and 36 to alanine (lanes 7 and 8), IκBα with amino acids 1 to 53 deleted (lanes 9 and 10), or IκBα with amino acids 139 to 317 deleted (lanes 11 and 12). The kinase assays were performed in the absence (−) or presence (+) of sheared salmon sperm DNA (0.5 μg), as indicated. (B) GST fusion proteins containing either wild-type IκBα (lanes 1 and 2), IκBα (amino acids [aa] 1 to 275) with the majority of the PEST domain deleted (lanes 3 and 4), full-length IκBα with a mutation of threonine residue 291 to alanine (lanes 5 and 6), IκBα with mutations of serine residue 283 and threonine residues 291 and 299 to alanine (lanes 7 and 8), or IκBα with threonine residues 273, 291, and 299 and serine residue 283 changed to alanine (lanes 9 and 10) were analyzed in kinase assays in either the absence (−) or presence (+) of double-stranded DNA and purified DNA-PK. (C) GST fusion proteins containing either wild-type IκBα (lane 1), full-length IκBα with serine residues 32 and 36 changed to alanine (lane 2), IκBα (aa 1 to 138) with a deletion of aa 139 to 317 (lane 3), this same construct with serine residues 32 and 36 mutated to alanine (lane 4), IκBα (aa 1 to 138) with serine residue 32 changed to alanine (lane 5), IκBα (aa 1 to 138) with serine residue 36 changed to alanine (lane 6), IκBα (aa 1 to 138) with tyrosine residue 42 changed to phenylalanine (lane 7), and IκBα (aa 1 to 138) with mutations of serine residues 32 and 36 to alanine and tyrosine 42 to phenylalanine (lane 8) were analyzed in kinase assays in the presence of double-stranded DNA and purified DNA-PK. (D) GST fusion proteins containing wild-type IκBβ (lane 1), this same construct with serine residues 19 and 23 mutated to alanine (lane 2), a truncated IκBβ construct (aa 1 to 305) (lane 3), or a truncated IκBβ construct with serine residues 19 and 23 changed to alanine (lane 4) were also analyzed in kinase assays with double-stranded DNA and purified DNA-PK.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Purification, Construct, Mutagenesis

    Constitutive and inducible cellular kinases that phosphorylate the amino terminus of IκBα. S100 extract was prepared from HeLa cells that were either untreated (A) or treated with 50 ng of phorbol ester (TPA) per ml (B) for 1 h. The extract was fractionated on a phosphocellulose column, washed with 0.1 M KCl, and eluted with 0.3 M KCl. Each of these extracts was concentrated on a Q-Sepharose column and then fractionated on a Superdex 200 column. The column fractions from the Superdex 200 column were then assayed for their ability to bind and phosphorylate a GST-IκBα fusion protein truncated at amino acid 138. The positions of the molecular weight markers in the Superdex 200 column fractions are indicated. (C) Fraction 19 from untreated HeLa S100 extract that eluted from the mono Q column was assayed for its ability to phosphorylate 0.5 μg of either GST (lane 1), a GST-IκBα fusion protein truncated at amino acid 138 that contained wild-type or mutant sequences at serine residues 32 and 36 (lanes 2 and 3, respectively), and a GST-IκBα fusion protein truncated at amino acid 53 containing wild-type or mutant sequences at serine residues 32 and 36 (lanes 4 and 5, respectively). Similar results were obtained with TPA-treated extract.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Constitutive and inducible cellular kinases that phosphorylate the amino terminus of IκBα. S100 extract was prepared from HeLa cells that were either untreated (A) or treated with 50 ng of phorbol ester (TPA) per ml (B) for 1 h. The extract was fractionated on a phosphocellulose column, washed with 0.1 M KCl, and eluted with 0.3 M KCl. Each of these extracts was concentrated on a Q-Sepharose column and then fractionated on a Superdex 200 column. The column fractions from the Superdex 200 column were then assayed for their ability to bind and phosphorylate a GST-IκBα fusion protein truncated at amino acid 138. The positions of the molecular weight markers in the Superdex 200 column fractions are indicated. (C) Fraction 19 from untreated HeLa S100 extract that eluted from the mono Q column was assayed for its ability to phosphorylate 0.5 μg of either GST (lane 1), a GST-IκBα fusion protein truncated at amino acid 138 that contained wild-type or mutant sequences at serine residues 32 and 36 (lanes 2 and 3, respectively), and a GST-IκBα fusion protein truncated at amino acid 53 containing wild-type or mutant sequences at serine residues 32 and 36 (lanes 4 and 5, respectively). Similar results were obtained with TPA-treated extract.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Molecular Weight, Mutagenesis

    Fractionation of IκBα kinase activity. A schematic of the purification scheme used to purify cellular kinases that phosphorylate the amino terminus of IκBα is shown. S100 extract was obtained from untreated HeLa cells and fractionated on a phosphocellulose column washed with buffer containing 0.1 M KCl and eluted with 0.3 M KCl. This fraction was applied to a Q-Sepharose (Q-seph) column and eluted with 1.0 M KCl, followed by fractionation on a Superdex 200 column. Proteins with kinase activity for the 138 amino-terminal residues of IκBα were then fractionated on heparin agarose, mono Q FPLC, and mono S FPLC and eluted with KCl gradients as indicated. FT, column flowthrough.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Fractionation of IκBα kinase activity. A schematic of the purification scheme used to purify cellular kinases that phosphorylate the amino terminus of IκBα is shown. S100 extract was obtained from untreated HeLa cells and fractionated on a phosphocellulose column washed with buffer containing 0.1 M KCl and eluted with 0.3 M KCl. This fraction was applied to a Q-Sepharose (Q-seph) column and eluted with 1.0 M KCl, followed by fractionation on a Superdex 200 column. Proteins with kinase activity for the 138 amino-terminal residues of IκBα were then fractionated on heparin agarose, mono Q FPLC, and mono S FPLC and eluted with KCl gradients as indicated. FT, column flowthrough.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Fractionation, Activity Assay, Purification, Fast Protein Liquid Chromatography

    Purified DNA-dependent protein kinase phosphorylates the amino terminus of IκBα. (A) Fractions (17 to 25) from the final mono S FPLC column were subjected to SDS-PAGE and Coomassie staining. (B) Kinase assays were also performed with these column fractions by using a GST-IκBα fusion protein extending from amino acids 1 to 138. (C) These same column fractions were also analyzed by Western blotting using polyclonal goat antibody directed against the DNA-dependent protein kinase catalytic subunit.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Purified DNA-dependent protein kinase phosphorylates the amino terminus of IκBα. (A) Fractions (17 to 25) from the final mono S FPLC column were subjected to SDS-PAGE and Coomassie staining. (B) Kinase assays were also performed with these column fractions by using a GST-IκBα fusion protein extending from amino acids 1 to 138. (C) These same column fractions were also analyzed by Western blotting using polyclonal goat antibody directed against the DNA-dependent protein kinase catalytic subunit.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Purification, Fast Protein Liquid Chromatography, SDS Page, Staining, Western Blot

    Antibody directed against DNA-PK immunoprecipitates IκBα kinase activity. (A) Full-length GST-IκBα (lane 1), GST-IκBβ (lane 2), and α-casein (lane 3) were used in in vitro kinase assays with mono S column fractions immunoprecipitated with either the specific monoclonal antibody 42-26 directed against DNA-PKcs (lanes 1 to 3) or the unrelated flag monoclonal antibody (lanes 4 to 6), followed by SDS-PAGE and autoradiography. (B) In vitro kinase assays were performed with GST-IκBα truncated at amino acid 138 and the mono S column fractions containing DNA-PK (lane 1) in the presence of 50 or 250 nM wortmannin (Wort) (lanes 2 and 3, respectively) or 50 or 250 nM rapamycin (Rap) (lanes 4 and 5, respectively). (C) Immunoprecipitation of DNA-PK activity was performed with monoclonal antibodies (42-26 and 25-4) directed against DNA-PKcs. Cytoplasmic extracts prepared by either the Dignam method (lanes 1 to 3) or the rapid-lysis method (lanes 4 to 6) were immunoprecipitated with these antibodies and analyzed in in vitro kinase assays without substrate (lanes 1 and 4), with GST (lanes 2 and 5), or with GST-IκBα (lanes 3 and 6).

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Antibody directed against DNA-PK immunoprecipitates IκBα kinase activity. (A) Full-length GST-IκBα (lane 1), GST-IκBβ (lane 2), and α-casein (lane 3) were used in in vitro kinase assays with mono S column fractions immunoprecipitated with either the specific monoclonal antibody 42-26 directed against DNA-PKcs (lanes 1 to 3) or the unrelated flag monoclonal antibody (lanes 4 to 6), followed by SDS-PAGE and autoradiography. (B) In vitro kinase assays were performed with GST-IκBα truncated at amino acid 138 and the mono S column fractions containing DNA-PK (lane 1) in the presence of 50 or 250 nM wortmannin (Wort) (lanes 2 and 3, respectively) or 50 or 250 nM rapamycin (Rap) (lanes 4 and 5, respectively). (C) Immunoprecipitation of DNA-PK activity was performed with monoclonal antibodies (42-26 and 25-4) directed against DNA-PKcs. Cytoplasmic extracts prepared by either the Dignam method (lanes 1 to 3) or the rapid-lysis method (lanes 4 to 6) were immunoprecipitated with these antibodies and analyzed in in vitro kinase assays without substrate (lanes 1 and 4), with GST (lanes 2 and 5), or with GST-IκBα (lanes 3 and 6).

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Activity Assay, In Vitro, Immunoprecipitation, SDS Page, Autoradiography, Lysis

    DNA-PK associates with IκBα. (A) HeLa S100 extract was incubated with a variety of GST-IκBα fusion proteins bound to glutathione agarose beads, followed by Western blot analysis with DNA-PKcs antibody. HeLa S100 extract with 50% of the input shown (lane 1) was incubated with either GST alone (lane 2), GST–wild-type IκBα (lane 3), GST-IκBα with mutations at serine residues 32 and 36 (la ne 4), GST-IκBα with its amino terminal 53 amino acids deleted (lane 5), GST-IκBα containing the amino-terminal 138 amino acids of IκBα (lane 6), or GST-IκBα containing the amino terminal 53 amino acids of IκBα (lane 7). Western blot analysis was performed with a goat polyclonal antibody directed against DNA-PKcs. (B) The GST-IκBα fusion proteins used in panel A were subjected to SDS-PAGE and Western blot analysis with antibody directed against GST.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: DNA-PK associates with IκBα. (A) HeLa S100 extract was incubated with a variety of GST-IκBα fusion proteins bound to glutathione agarose beads, followed by Western blot analysis with DNA-PKcs antibody. HeLa S100 extract with 50% of the input shown (lane 1) was incubated with either GST alone (lane 2), GST–wild-type IκBα (lane 3), GST-IκBα with mutations at serine residues 32 and 36 (la ne 4), GST-IκBα with its amino terminal 53 amino acids deleted (lane 5), GST-IκBα containing the amino-terminal 138 amino acids of IκBα (lane 6), or GST-IκBα containing the amino terminal 53 amino acids of IκBα (lane 7). Western blot analysis was performed with a goat polyclonal antibody directed against DNA-PKcs. (B) The GST-IκBα fusion proteins used in panel A were subjected to SDS-PAGE and Western blot analysis with antibody directed against GST.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Incubation, Western Blot, SDS Page

    Schematic structure of IκBα and IκBβ fusion constructs. (A) GST fusion proteins were constructed with wild-type IκBα (lane 1), IκBα with mutations of serine residues 32 and 36 to alanine (lane 2), IκBα with mutations of serine residue 283 and threonine residues 291 and 299 to alanine (lane 3), IκBα with mutations of threonine residues 273, 291, and 299 and serine residue 283 to alanine (lane 4), IκBα with residues 244 to 317 deleted (lane 5), IκBα with residues 275 to 317 deleted (lane 6), IκBα with its amino-terminal 53 amino acids deleted (lane 7), IκBα truncated at amino acid 138 (lane 8), this construct with mutations of serine residues 32 and 36 to alanine (lane 9), serine residue 32 to alanine (lane 10), serine residue 36 to alanine (lane 11), tyrosine residue 42 to phenylalanine (lane 12), or serine residues 32 and 36 to alanine and tyrosine residue 42 to phenylalanine (lane 13). (B) GST fusion proteins were constructed with the full-length IκBβ (lane 1), this same construct having mutations of serine residues 19 and 23 to alanine (lane 2), IκBβ truncated at amino acid 305 (lane 3), and this protein with serine residues 19 and 23 changed to alanine (lane 4). The positions of the ankyrin repeats, the PEST domain, and serine and/or tyrosine residues that were mutated are shown.

    Journal: Molecular and Cellular Biology

    Article Title: DNA-Dependent Protein Kinase Phosphorylation of I?B? and I?B? Regulates NF-?B DNA Binding Properties

    doi:

    Figure Lengend Snippet: Schematic structure of IκBα and IκBβ fusion constructs. (A) GST fusion proteins were constructed with wild-type IκBα (lane 1), IκBα with mutations of serine residues 32 and 36 to alanine (lane 2), IκBα with mutations of serine residue 283 and threonine residues 291 and 299 to alanine (lane 3), IκBα with mutations of threonine residues 273, 291, and 299 and serine residue 283 to alanine (lane 4), IκBα with residues 244 to 317 deleted (lane 5), IκBα with residues 275 to 317 deleted (lane 6), IκBα with its amino-terminal 53 amino acids deleted (lane 7), IκBα truncated at amino acid 138 (lane 8), this construct with mutations of serine residues 32 and 36 to alanine (lane 9), serine residue 32 to alanine (lane 10), serine residue 36 to alanine (lane 11), tyrosine residue 42 to phenylalanine (lane 12), or serine residues 32 and 36 to alanine and tyrosine residue 42 to phenylalanine (lane 13). (B) GST fusion proteins were constructed with the full-length IκBβ (lane 1), this same construct having mutations of serine residues 19 and 23 to alanine (lane 2), IκBβ truncated at amino acid 305 (lane 3), and this protein with serine residues 19 and 23 changed to alanine (lane 4). The positions of the ankyrin repeats, the PEST domain, and serine and/or tyrosine residues that were mutated are shown.

    Article Snippet: The cells were washed twice with PBS, blocked for 30 min in PBS containing 0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBα (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit polyclonal antibody directed against a carboxy-terminal peptide of IκBβ p65 (C-20; Santa Cruz Biotechnology, Inc.).

    Techniques: Construct

    Specificity of the interaction of tyrosine-phosphorylated IκB-α with the regulatory p85α subunit of PI3-kinase. ( A ) Interaction of tyrosine-phosphorylated IκB-α with the C-terminal SH2 domain of p85α. Two μg of GST-C-SH2 agarose conjugate (lanes 1 and 2) were incubated with whole-cell extracts from control (−) or 200 μM pV-treated (+) Jurkat T cells. The binding of IκB-α to the GST proteins was detected by Western blotting. The analysis of total cell extract by Western blotting for IκB-α is shown in lanes 3 and 4. The positions of IκB-α and its tyrosine-phosphorylated form, as well as that of the GST fusion protein, are indicated by arrows. ( B ) Specificity of interaction of tyrosine-phosphorylated IκB-α with p85α. Whole-cell extracts from control (−) and pV-treated (+) Jurkat T cells were incubated with 6 μM of the indicated peptides. Two μg of GST (lanes 1 and 2) or GST-p85α (lanes 3–10) agarose conjugates were added and the presence of IκB-α in the complexes was analyzed by Western blotting. The position of tyrosine-phosphorylated IκB-α is indicated.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-?B activation

    doi:

    Figure Lengend Snippet: Specificity of the interaction of tyrosine-phosphorylated IκB-α with the regulatory p85α subunit of PI3-kinase. ( A ) Interaction of tyrosine-phosphorylated IκB-α with the C-terminal SH2 domain of p85α. Two μg of GST-C-SH2 agarose conjugate (lanes 1 and 2) were incubated with whole-cell extracts from control (−) or 200 μM pV-treated (+) Jurkat T cells. The binding of IκB-α to the GST proteins was detected by Western blotting. The analysis of total cell extract by Western blotting for IκB-α is shown in lanes 3 and 4. The positions of IκB-α and its tyrosine-phosphorylated form, as well as that of the GST fusion protein, are indicated by arrows. ( B ) Specificity of interaction of tyrosine-phosphorylated IκB-α with p85α. Whole-cell extracts from control (−) and pV-treated (+) Jurkat T cells were incubated with 6 μM of the indicated peptides. Two μg of GST (lanes 1 and 2) or GST-p85α (lanes 3–10) agarose conjugates were added and the presence of IκB-α in the complexes was analyzed by Western blotting. The position of tyrosine-phosphorylated IκB-α is indicated.

    Article Snippet: Anti–IκB-α and anti-p110β polyclonal antisera were obtained from Santa Cruz Biotechnology.

    Techniques: Incubation, Binding Assay, Western Blot

    Wortmannin does not perturb tyrosine phosphorylation of IκB-α and PI3-kinase heterodimer formation. ( A ) Effect of wortmannin on pV-mediated tyrosine phosphorylation of IκB-α. Jurkat T cells were pretreated with 200 nM of wortmannin (lanes 3 and 4) and induced with 200 μM pV (lanes 2 and 3) for 1 h. IκB-α phosphorylation status was analyzed by Western blotting. The position of IκB-α and tyrosine phosphorylated IκB-α is indicated. ( B ) Effect of wortmannin on PI3-kinase heterodimer formation. Jurkat T cells were pretreated with 200 nM of wortmannin (lanes 3 and 6) and were subsequently induced with 1 mM pV (lanes 2, 3, 5, 6) for 1 h. Cytosolic extracts were prepared and immunoprecipitated with anti-p85α (lanes 1–3) or anti-p110β (lanes 4–6). The presence of p85α ( Upper ) and p110β ( Lower ) in the immunocomplexes was analyzed by Western blotting. The positions of p85α and p110β are indicated by arrows. ( C ) pV induces PI3-kinase activity as measured by Akt serine 473 phosphorylation. Jurkat cells were pretreated (lanes 4–6) or not (lanes 1–3) with 100 nM of wortmannin and induced with 400 μM pV for 0 (lanes 1 and 4), 15 (lanes 2 and 5), or 30 min (lanes 3 and 6). The phosphorylation status of Akt was analyzed by Western blotting with an antibody specific for phosphorylated Akt ( Upper ). Total levels of Akt were measured with an anti-Akt antibody ( Lower ). The positions of phosphoserine 473-Akt and Akt are indicated by arrows.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-?B activation

    doi:

    Figure Lengend Snippet: Wortmannin does not perturb tyrosine phosphorylation of IκB-α and PI3-kinase heterodimer formation. ( A ) Effect of wortmannin on pV-mediated tyrosine phosphorylation of IκB-α. Jurkat T cells were pretreated with 200 nM of wortmannin (lanes 3 and 4) and induced with 200 μM pV (lanes 2 and 3) for 1 h. IκB-α phosphorylation status was analyzed by Western blotting. The position of IκB-α and tyrosine phosphorylated IκB-α is indicated. ( B ) Effect of wortmannin on PI3-kinase heterodimer formation. Jurkat T cells were pretreated with 200 nM of wortmannin (lanes 3 and 6) and were subsequently induced with 1 mM pV (lanes 2, 3, 5, 6) for 1 h. Cytosolic extracts were prepared and immunoprecipitated with anti-p85α (lanes 1–3) or anti-p110β (lanes 4–6). The presence of p85α ( Upper ) and p110β ( Lower ) in the immunocomplexes was analyzed by Western blotting. The positions of p85α and p110β are indicated by arrows. ( C ) pV induces PI3-kinase activity as measured by Akt serine 473 phosphorylation. Jurkat cells were pretreated (lanes 4–6) or not (lanes 1–3) with 100 nM of wortmannin and induced with 400 μM pV for 0 (lanes 1 and 4), 15 (lanes 2 and 5), or 30 min (lanes 3 and 6). The phosphorylation status of Akt was analyzed by Western blotting with an antibody specific for phosphorylated Akt ( Upper ). Total levels of Akt were measured with an anti-Akt antibody ( Lower ). The positions of phosphoserine 473-Akt and Akt are indicated by arrows.

    Article Snippet: Anti–IκB-α and anti-p110β polyclonal antisera were obtained from Santa Cruz Biotechnology.

    Techniques: Western Blot, Immunoprecipitation, Activity Assay

    Proteins interacting with a tyrosine-phosphorylated IκB-α peptide. ( A ) Affinity purification. The sequence of the peptides used for the affinity purification from 293 cell extracts is shown on top. Amino acid residues 37–48 of IκB-α were fused to four glycine residues and a Flag epitope at the C terminus. Tyrosine 42 was either phosphorylated (Y42-P) or not (Y42). Proteins interacting with the nonphosphorylated peptide (lane 1) or the phosphorylated peptide (lane 2) were analyzed by SDS polyacrylamide gel electrophoresis and Coomassie blue staining. The position of two molecular mass markers is indicated on the left; the position of three proteins interacting only with the tyrosine-phosphorylated peptide is indicated on the right. Protein sequences of two tryptic peptides obtained from the 85-kDa band are shown underlined. They are found within the indicated regions of human PI3-kinase regulatory p85α subunit. ( B ) Both PI3-kinase subunits bind to the IκB-α peptide. Proteins from Jurkat T cells binding to the nonphosphorylated peptide (lanes 1 and 3) or tyrosine-phosphorylated IκB-α peptide (lanes 2 and 4) were analyzed by Western blotting using an antibody directed against the regulatory p85α subunit of PI3-kinase (lanes 1 and 2) or the catalytic p110β subunit of PI3-kinase (lanes 3 and 4). The positions of p85α and p110β are indicated by arrows on the right; the positions of four molecular mass markers on the left. ( C ) Interaction of endogenous p85α with endogenous IκB-α on pV treatment. Jurkat T cells (1.5 × 10 6 ) pretreated with 400 nM wortmannin were induced with 400 μM pV for 0 (lanes 1 and 5), 5 (lanes 2 and 6), 15 (lanes 3 and 7), and 30 min (lanes 4 and 8). Cytosolic extracts were prepared and immunoprecipitated with anti-p85α (lanes 1–4) or anti-IκB-α (lanes 5–8). The presence of p85α in the complexes was detected by Western blotting. The position of p85α is indicated by an arrow.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-?B activation

    doi:

    Figure Lengend Snippet: Proteins interacting with a tyrosine-phosphorylated IκB-α peptide. ( A ) Affinity purification. The sequence of the peptides used for the affinity purification from 293 cell extracts is shown on top. Amino acid residues 37–48 of IκB-α were fused to four glycine residues and a Flag epitope at the C terminus. Tyrosine 42 was either phosphorylated (Y42-P) or not (Y42). Proteins interacting with the nonphosphorylated peptide (lane 1) or the phosphorylated peptide (lane 2) were analyzed by SDS polyacrylamide gel electrophoresis and Coomassie blue staining. The position of two molecular mass markers is indicated on the left; the position of three proteins interacting only with the tyrosine-phosphorylated peptide is indicated on the right. Protein sequences of two tryptic peptides obtained from the 85-kDa band are shown underlined. They are found within the indicated regions of human PI3-kinase regulatory p85α subunit. ( B ) Both PI3-kinase subunits bind to the IκB-α peptide. Proteins from Jurkat T cells binding to the nonphosphorylated peptide (lanes 1 and 3) or tyrosine-phosphorylated IκB-α peptide (lanes 2 and 4) were analyzed by Western blotting using an antibody directed against the regulatory p85α subunit of PI3-kinase (lanes 1 and 2) or the catalytic p110β subunit of PI3-kinase (lanes 3 and 4). The positions of p85α and p110β are indicated by arrows on the right; the positions of four molecular mass markers on the left. ( C ) Interaction of endogenous p85α with endogenous IκB-α on pV treatment. Jurkat T cells (1.5 × 10 6 ) pretreated with 400 nM wortmannin were induced with 400 μM pV for 0 (lanes 1 and 5), 5 (lanes 2 and 6), 15 (lanes 3 and 7), and 30 min (lanes 4 and 8). Cytosolic extracts were prepared and immunoprecipitated with anti-p85α (lanes 1–4) or anti-IκB-α (lanes 5–8). The presence of p85α in the complexes was detected by Western blotting. The position of p85α is indicated by an arrow.

    Article Snippet: Anti–IκB-α and anti-p110β polyclonal antisera were obtained from Santa Cruz Biotechnology.

    Techniques: Affinity Purification, Sequencing, FLAG-tag, Polyacrylamide Gel Electrophoresis, Staining, Binding Assay, Western Blot, Immunoprecipitation

    The effect of TNFα on pV-induced events. ( A ) Protection of tyrosine-phosphorylated IκB-α from degradation through the TNFα pathway. Jurkat T cells were pretreated (lanes 1–4) or not (lanes 5–8) with 10 μg⋅ml −1 cycloheximide for 30 min, treated with 1 mM pV (lanes 2, 3, 6 and 7) and 10 min later induced with 50 ng⋅ml −1 TNFα (lanes 3, 4, 7 and 8) for 1 h. Whole-cell extracts were prepared and IκB-α was detected by Western blotting. The positions of IκB-α and its tyrosine-phosphorylated form are indicated. ( B ) pV does not impair TNFα signaling. Jurkat cells were transiently transfected with a plasmid containing a luciferase reporter gene driven by three repeats of the HIV type 1 (HIV-1) κB enhancer. Forty-eight hours after transfection, cells were treated with 1 mM pV and 1 h later induced with 50 ng⋅ml −1 TNFα for 5 h. Luciferase activities were measured and normalized on the basis of β-galactosidase expression from cotransfected pRSV-β-galactosidase. The values shown are averages (mean + SEM) of one representative experiment in which each transfection was performed in duplicate.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-?B activation

    doi:

    Figure Lengend Snippet: The effect of TNFα on pV-induced events. ( A ) Protection of tyrosine-phosphorylated IκB-α from degradation through the TNFα pathway. Jurkat T cells were pretreated (lanes 1–4) or not (lanes 5–8) with 10 μg⋅ml −1 cycloheximide for 30 min, treated with 1 mM pV (lanes 2, 3, 6 and 7) and 10 min later induced with 50 ng⋅ml −1 TNFα (lanes 3, 4, 7 and 8) for 1 h. Whole-cell extracts were prepared and IκB-α was detected by Western blotting. The positions of IκB-α and its tyrosine-phosphorylated form are indicated. ( B ) pV does not impair TNFα signaling. Jurkat cells were transiently transfected with a plasmid containing a luciferase reporter gene driven by three repeats of the HIV type 1 (HIV-1) κB enhancer. Forty-eight hours after transfection, cells were treated with 1 mM pV and 1 h later induced with 50 ng⋅ml −1 TNFα for 5 h. Luciferase activities were measured and normalized on the basis of β-galactosidase expression from cotransfected pRSV-β-galactosidase. The values shown are averages (mean + SEM) of one representative experiment in which each transfection was performed in duplicate.

    Article Snippet: Anti–IκB-α and anti-p110β polyclonal antisera were obtained from Santa Cruz Biotechnology.

    Techniques: Western Blot, Transfection, Plasmid Preparation, Luciferase, Expressing

    Agent based modelling including binding and release of cytoskeletal IκBα. A. Flowchart summarising the activation cascade and downstream effects incorporated into the agent based model of the NF-κB signalling pathway. B. Representation of localisation and movement of selected agents within the agent based model. C, D, E. Simulations of un-stimulated levels show accumulation of IκBα on the cytoskeleton with dissociation from NF-κB only (C) but a steady state when IκBα dissociates from neither (D) or both (E) cytoskeleton and NF-κB. F-H. Simulations of responses to IL-1 stimulation in the model with dissociation from neither cytoskeleton nor NF-κB. I-K Simulations of response to IL-1 stimulation in the model with dissociation from both cytoskeleton and NF-κB. Graphs show the average of 3 simulations for each condition. p

    Journal: PLoS ONE

    Article Title: Computational Modelling of NF-κB Activation by IL-1RI and Its Co-Receptor TILRR, Predicts a Role for Cytoskeletal Sequestration of IκBα in Inflammatory Signalling

    doi: 10.1371/journal.pone.0129888

    Figure Lengend Snippet: Agent based modelling including binding and release of cytoskeletal IκBα. A. Flowchart summarising the activation cascade and downstream effects incorporated into the agent based model of the NF-κB signalling pathway. B. Representation of localisation and movement of selected agents within the agent based model. C, D, E. Simulations of un-stimulated levels show accumulation of IκBα on the cytoskeleton with dissociation from NF-κB only (C) but a steady state when IκBα dissociates from neither (D) or both (E) cytoskeleton and NF-κB. F-H. Simulations of responses to IL-1 stimulation in the model with dissociation from neither cytoskeleton nor NF-κB. I-K Simulations of response to IL-1 stimulation in the model with dissociation from both cytoskeleton and NF-κB. Graphs show the average of 3 simulations for each condition. p

    Article Snippet: Membranes were incubated with Milk (5%, Tris Buffered Saline Tween, TBST) and with an anti-IκBα antibody (1:1000; C21 rabbit polyclonal, O/N, 4°C, Santa Cruz), an anti-phospho-IκBα antibody (1/1000, Cell Signalling), an anti-spectrin antibody (1/1000) and with anti-β-actin (1/1000), washed in 1x TBST, incubated with IRDye 800CW Donkey anti-Rabbit (1:10,000, 1 hr; LI-COR Biosciences), washed, and developed using LI-COR Odyssey.

    Techniques: Binding Assay, Activation Assay

    The model accurately reproduces activation of inflammatory and anti-apoptotic signals, controlled through IL-1RI and TILRR. A. TILRR siRNA decreases IκBα degradation in HeLa cells transfected with IκBα-EGFP (7μg/10 6 cells) alone or in the presence of random (♦) or TILRR-specific (■) siRNA and stimulated with IL-1 (1 nM). Graph show continuous readings from three experiments including 55 cells, p

    Journal: PLoS ONE

    Article Title: Computational Modelling of NF-κB Activation by IL-1RI and Its Co-Receptor TILRR, Predicts a Role for Cytoskeletal Sequestration of IκBα in Inflammatory Signalling

    doi: 10.1371/journal.pone.0129888

    Figure Lengend Snippet: The model accurately reproduces activation of inflammatory and anti-apoptotic signals, controlled through IL-1RI and TILRR. A. TILRR siRNA decreases IκBα degradation in HeLa cells transfected with IκBα-EGFP (7μg/10 6 cells) alone or in the presence of random (♦) or TILRR-specific (■) siRNA and stimulated with IL-1 (1 nM). Graph show continuous readings from three experiments including 55 cells, p

    Article Snippet: Membranes were incubated with Milk (5%, Tris Buffered Saline Tween, TBST) and with an anti-IκBα antibody (1:1000; C21 rabbit polyclonal, O/N, 4°C, Santa Cruz), an anti-phospho-IκBα antibody (1/1000, Cell Signalling), an anti-spectrin antibody (1/1000) and with anti-β-actin (1/1000), washed in 1x TBST, incubated with IRDye 800CW Donkey anti-Rabbit (1:10,000, 1 hr; LI-COR Biosciences), washed, and developed using LI-COR Odyssey.

    Techniques: Activation Assay, Transfection

    IκBα interaction with cytoskeletal proteins. A. Space-filling representation of the predicted binding interaction of IκBα with cytoskeletal proteins spectrin and actin. Two orientations of the complex are shown to illustrate binding interactions between the three molecules. β-spectrin is shown in blue, actin in red and IκBα in yellow. B. HeLa cells were transfected with IκBα-EGFP together with a non-targeting siRNA or TILRR siRNA. Cell lysates were immunoprecipitated with an anti-spectrin antibody, and levels of spectrin and of spectrin-associated IκBα determined by immunoblotting, as described in material and methods. C. Quantitation of spectrin associated IκBα-EGFP from western blots of cells transfected with random siRNA (control) or TILRR siRNA, and stimulated with IL-1β (1nM) over time, as in B. Data are presented as fraction of levels in un-stimulated samples (t = 0) and expressed relative to loading control (Mean ±SEM, n = 5, p

    Journal: PLoS ONE

    Article Title: Computational Modelling of NF-κB Activation by IL-1RI and Its Co-Receptor TILRR, Predicts a Role for Cytoskeletal Sequestration of IκBα in Inflammatory Signalling

    doi: 10.1371/journal.pone.0129888

    Figure Lengend Snippet: IκBα interaction with cytoskeletal proteins. A. Space-filling representation of the predicted binding interaction of IκBα with cytoskeletal proteins spectrin and actin. Two orientations of the complex are shown to illustrate binding interactions between the three molecules. β-spectrin is shown in blue, actin in red and IκBα in yellow. B. HeLa cells were transfected with IκBα-EGFP together with a non-targeting siRNA or TILRR siRNA. Cell lysates were immunoprecipitated with an anti-spectrin antibody, and levels of spectrin and of spectrin-associated IκBα determined by immunoblotting, as described in material and methods. C. Quantitation of spectrin associated IκBα-EGFP from western blots of cells transfected with random siRNA (control) or TILRR siRNA, and stimulated with IL-1β (1nM) over time, as in B. Data are presented as fraction of levels in un-stimulated samples (t = 0) and expressed relative to loading control (Mean ±SEM, n = 5, p

    Article Snippet: Membranes were incubated with Milk (5%, Tris Buffered Saline Tween, TBST) and with an anti-IκBα antibody (1:1000; C21 rabbit polyclonal, O/N, 4°C, Santa Cruz), an anti-phospho-IκBα antibody (1/1000, Cell Signalling), an anti-spectrin antibody (1/1000) and with anti-β-actin (1/1000), washed in 1x TBST, incubated with IRDye 800CW Donkey anti-Rabbit (1:10,000, 1 hr; LI-COR Biosciences), washed, and developed using LI-COR Odyssey.

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Quantitation Assay, Western Blot

    Cytoskeletal binding and release of IκBα controls NF-κB signalling and gene induction. A. Simulated IL-8 gene activity kinetics at varying IL-1 pathway stimulation levels, in the presence (Red) and absence (Blue) of cytoskeletal binding and release of IκBα. Simulations show that disabling cytoskeletal binding of the inhibitor abrogates activation at low levels of stimulation, causes delayed responses at medium stimulation, and results in a slightly delayed but significantly increased activation at high levels of stimulus. P

    Journal: PLoS ONE

    Article Title: Computational Modelling of NF-κB Activation by IL-1RI and Its Co-Receptor TILRR, Predicts a Role for Cytoskeletal Sequestration of IκBα in Inflammatory Signalling

    doi: 10.1371/journal.pone.0129888

    Figure Lengend Snippet: Cytoskeletal binding and release of IκBα controls NF-κB signalling and gene induction. A. Simulated IL-8 gene activity kinetics at varying IL-1 pathway stimulation levels, in the presence (Red) and absence (Blue) of cytoskeletal binding and release of IκBα. Simulations show that disabling cytoskeletal binding of the inhibitor abrogates activation at low levels of stimulation, causes delayed responses at medium stimulation, and results in a slightly delayed but significantly increased activation at high levels of stimulus. P

    Article Snippet: Membranes were incubated with Milk (5%, Tris Buffered Saline Tween, TBST) and with an anti-IκBα antibody (1:1000; C21 rabbit polyclonal, O/N, 4°C, Santa Cruz), an anti-phospho-IκBα antibody (1/1000, Cell Signalling), an anti-spectrin antibody (1/1000) and with anti-β-actin (1/1000), washed in 1x TBST, incubated with IRDye 800CW Donkey anti-Rabbit (1:10,000, 1 hr; LI-COR Biosciences), washed, and developed using LI-COR Odyssey.

    Techniques: Binding Assay, Activity Assay, Activation Assay

    IL-1-induced release of IκBα from the cytoskeleton is characterized by high Kon /Koff rates. A, B. Simulation of levels of IκBα bound to the cytoskeleton during IL-1β stimulation under various settings. Release from the cytoskeleton was governed by Kon and Koff rates, adjusted to be high (A) or low (B) at the same Ka, with 2/3 of IκBα bound to the cytoskeleton at time = 0. Inhibitor release was set at 3 levels passive release only (Red), or release of 40–50% (Green) or of 90–100% (Blue). The model including high Kon and Koff rates fits best with in vitro data seen in Fig 2C . A. p

    Journal: PLoS ONE

    Article Title: Computational Modelling of NF-κB Activation by IL-1RI and Its Co-Receptor TILRR, Predicts a Role for Cytoskeletal Sequestration of IκBα in Inflammatory Signalling

    doi: 10.1371/journal.pone.0129888

    Figure Lengend Snippet: IL-1-induced release of IκBα from the cytoskeleton is characterized by high Kon /Koff rates. A, B. Simulation of levels of IκBα bound to the cytoskeleton during IL-1β stimulation under various settings. Release from the cytoskeleton was governed by Kon and Koff rates, adjusted to be high (A) or low (B) at the same Ka, with 2/3 of IκBα bound to the cytoskeleton at time = 0. Inhibitor release was set at 3 levels passive release only (Red), or release of 40–50% (Green) or of 90–100% (Blue). The model including high Kon and Koff rates fits best with in vitro data seen in Fig 2C . A. p

    Article Snippet: Membranes were incubated with Milk (5%, Tris Buffered Saline Tween, TBST) and with an anti-IκBα antibody (1:1000; C21 rabbit polyclonal, O/N, 4°C, Santa Cruz), an anti-phospho-IκBα antibody (1/1000, Cell Signalling), an anti-spectrin antibody (1/1000) and with anti-β-actin (1/1000), washed in 1x TBST, incubated with IRDye 800CW Donkey anti-Rabbit (1:10,000, 1 hr; LI-COR Biosciences), washed, and developed using LI-COR Odyssey.

    Techniques: In Vitro

    Quercetin inhibits ZD55-TRAIL induced NF-κB activation in HCC cells. Notes: HuH-7 cells were cultured with ZD55-TRAIL (2MOI), quercetin (10 μM), or ZD55-TRAIL (2MOI) plus quercetin (10 μM) for 48 h. Cell lysates were prepared and ( A ) Western blottingor ( B ) ELISA was performed to examine the changes in IκBα, p65, and p50 expression. GAPDH was used as the loading control. ( C ) Apoptosis-related proteins Bcl-2, FLIP, Bid and Bax were detected by Western blotting after treatment with ZD55-TRAIL (2MOI), quercetin (10 μM), or ZD55-TRAIL (2MOI) plus quercetin (10 μM) for 48 h. Differences in protein levels was determined by densitometry and expressed as percentages. Data are presented as mean ± SD and are representative of three separate experiments. (*Represents P

    Journal: Scientific Reports

    Article Title: Synergistic Anti-tumour Effects of Quercetin and Oncolytic Adenovirus expressing TRAIL in Human Hepatocellular Carcinoma

    doi: 10.1038/s41598-018-20213-7

    Figure Lengend Snippet: Quercetin inhibits ZD55-TRAIL induced NF-κB activation in HCC cells. Notes: HuH-7 cells were cultured with ZD55-TRAIL (2MOI), quercetin (10 μM), or ZD55-TRAIL (2MOI) plus quercetin (10 μM) for 48 h. Cell lysates were prepared and ( A ) Western blottingor ( B ) ELISA was performed to examine the changes in IκBα, p65, and p50 expression. GAPDH was used as the loading control. ( C ) Apoptosis-related proteins Bcl-2, FLIP, Bid and Bax were detected by Western blotting after treatment with ZD55-TRAIL (2MOI), quercetin (10 μM), or ZD55-TRAIL (2MOI) plus quercetin (10 μM) for 48 h. Differences in protein levels was determined by densitometry and expressed as percentages. Data are presented as mean ± SD and are representative of three separate experiments. (*Represents P

    Article Snippet: Adenovirus-5 E1A, Capase-9, Caspase-3, PARP and GAPDH primary antibodies were bought from Santa Cruz Biotechnology and those against p65, p60 and IκBα from Cell Signalling.

    Techniques: Activation Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

    TRAIL induces the phosphorylation of IκBα. ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). Cells stimulated with macrophage-conditioned medium (MaCM) were used as a positive control. The phosphorylation of IκBα was analyzed by Western blot. α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. ( B ) SGBS adipocytes on day 14 of adipogenic differentiation were treated for 2 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and nuclear extracts were prepared. DNA binding activity of NFκB was analyzed by electrophoretic mobility shift assay (EMSA). One representative experiment out of three performed experiments is presented. ( C ) SGBS adipocytes on day 7 of adipogenic differentiation were transfected with NFκB Firefly luciferase reporter vector and Renilla luciferase control reporter vector. On day 9, cells were treated for 24 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and luciferase activity was determined. Values are means and SEM of 3 different experiments. Unpaired Student´s t-test was used to test for statistical significance. ( D-H ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the IKK inhibitor SC-514 (100 μM). After 6 hours, the phosphorylation of IκBα was analyzed by Western blot ( D ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, the expression of IL-6 ( E ), IL-8 ( F ), MCP-1 ( G ) and CCL-20 ( H ) was assessed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p

    Journal: Scientific Reports

    Article Title: Trail (TNF-related apoptosis-inducing ligand) induces an inflammatory response in human adipocytes

    doi: 10.1038/s41598-017-05932-7

    Figure Lengend Snippet: TRAIL induces the phosphorylation of IκBα. ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). Cells stimulated with macrophage-conditioned medium (MaCM) were used as a positive control. The phosphorylation of IκBα was analyzed by Western blot. α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. ( B ) SGBS adipocytes on day 14 of adipogenic differentiation were treated for 2 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and nuclear extracts were prepared. DNA binding activity of NFκB was analyzed by electrophoretic mobility shift assay (EMSA). One representative experiment out of three performed experiments is presented. ( C ) SGBS adipocytes on day 7 of adipogenic differentiation were transfected with NFκB Firefly luciferase reporter vector and Renilla luciferase control reporter vector. On day 9, cells were treated for 24 hours with TRAIL (30 ng/ml), TNF-α (30 mg/ml) or vehicle and luciferase activity was determined. Values are means and SEM of 3 different experiments. Unpaired Student´s t-test was used to test for statistical significance. ( D-H ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the IKK inhibitor SC-514 (100 μM). After 6 hours, the phosphorylation of IκBα was analyzed by Western blot ( D ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, the expression of IL-6 ( E ), IL-8 ( F ), MCP-1 ( G ) and CCL-20 ( H ) was assessed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p

    Article Snippet: The following antibodies were used: rabbit anti-phospho AKT (S473), rabbit anti-AKT, mouse anti-phospho ERK1/2 (T202/Y204), rabbit anti-phopsho JNK (T183/Y185), mouse anti-phospho IκBα (S32/S36), rabbit anti-IκBα, rabbit anti-caspase-3 (Cell signaling, Cambridge, UK), mouse anti-JNK (BD, Heidelberg, Germany), mouse anti-caspase-8 (Alexis, Grünberg, Germany), rabbit anti-ERK1/2, mouse anti-β-actin (Sigma-Aldrich, Munich, Germany), mouse anti-caspase-8 (Enzo LifeSciences, Lörrach, Germany) and mouse anti-α-tubulin (Calbiochem/EMD Millipore, Darmstadt, Germany).

    Techniques: Isolation, Positive Control, Western Blot, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction

    TRAIL induces the phosphorylation of ERK1/2. ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). The phosphorylation of ERK1/2, JNK and AKT was determined by Western blot. ( B – F ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the MEK1/2 inhibitor PD-0325901 (100 nM). After 6 hours, the phosphorylation of ERK1/2 and IκBα was analyzed by Western blot ( B ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, IL-6 ( C ), IL-8 ( D ), MCP-1 ( E ) and CCL-20 ( F ) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p

    Journal: Scientific Reports

    Article Title: Trail (TNF-related apoptosis-inducing ligand) induces an inflammatory response in human adipocytes

    doi: 10.1038/s41598-017-05932-7

    Figure Lengend Snippet: TRAIL induces the phosphorylation of ERK1/2. ( A ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle and protein was isolated at different timepoints (1/4, 1/2, 1, 2 and 6 hours). The phosphorylation of ERK1/2, JNK and AKT was determined by Western blot. ( B – F ) SGBS adipocytes on day 14 of adipogenic differentiation were treated with TRAIL (30 ng/ml) or vehicle in the absence or presence of the MEK1/2 inhibitor PD-0325901 (100 nM). After 6 hours, the phosphorylation of ERK1/2 and IκBα was analyzed by Western blot ( B ). α-tubulin was used as a loading control. One representative blot out of three performed experiments is presented. Also, IL-6 ( C ), IL-8 ( D ), MCP-1 ( E ) and CCL-20 ( F ) was analyzed by qPCR. The mRNA levels were normalized to HPRT. Depicted are the means and SEM of 4 independent experiments. One-way ANOVA and Dunnett’s multiple comparison were used to test for statistical significance. *p

    Article Snippet: The following antibodies were used: rabbit anti-phospho AKT (S473), rabbit anti-AKT, mouse anti-phospho ERK1/2 (T202/Y204), rabbit anti-phopsho JNK (T183/Y185), mouse anti-phospho IκBα (S32/S36), rabbit anti-IκBα, rabbit anti-caspase-3 (Cell signaling, Cambridge, UK), mouse anti-JNK (BD, Heidelberg, Germany), mouse anti-caspase-8 (Alexis, Grünberg, Germany), rabbit anti-ERK1/2, mouse anti-β-actin (Sigma-Aldrich, Munich, Germany), mouse anti-caspase-8 (Enzo LifeSciences, Lörrach, Germany) and mouse anti-α-tubulin (Calbiochem/EMD Millipore, Darmstadt, Germany).

    Techniques: Isolation, Western Blot, Real-time Polymerase Chain Reaction

    SR reduces DMBA/TPA-induced inflammation in the skin of mice through inactivation of NF-κB in vivo . The skin was removed from mice treated under different conditions. Then, (A) RT-qPCR analysis was used to determine TNF-α, IL-1β, IL-18, IL-6, COX2 and TGF-β1 mRNA levels. (B) Western blot analysis was carried out to evaluate phosphorylated IκBα, IκBα and phosphorylated NF-κB in the skin tissue samples obtained from mice. (C) Representative images of skin after exposure to DMBA/TPA in the absence or presence of SR. The quantification of phosphorylated NF-κB was measured using immunofluorescence analysis. Data are presented as mean ± SEM (n=20). + P

    Journal: Oncology Reports

    Article Title: Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a mouse model through suppression of inflammation and promotion of apoptosis

    doi: 10.3892/or.2018.6381

    Figure Lengend Snippet: SR reduces DMBA/TPA-induced inflammation in the skin of mice through inactivation of NF-κB in vivo . The skin was removed from mice treated under different conditions. Then, (A) RT-qPCR analysis was used to determine TNF-α, IL-1β, IL-18, IL-6, COX2 and TGF-β1 mRNA levels. (B) Western blot analysis was carried out to evaluate phosphorylated IκBα, IκBα and phosphorylated NF-κB in the skin tissue samples obtained from mice. (C) Representative images of skin after exposure to DMBA/TPA in the absence or presence of SR. The quantification of phosphorylated NF-κB was measured using immunofluorescence analysis. Data are presented as mean ± SEM (n=20). + P

    Article Snippet: The primary antibodies used in our study included: anti-p21 (1:1,000, ab86696), anti-PUMA (1:1,000, ab9643), anti-Bax (1:1,000, ab32503), anti-p53 (1:1,000, ab131442), anti-caspase-3 (1:1,000, ab90437), anti-IκBα (1:1,000, ab32518), anti-p-IκBα (1:1,000, ab133462), anti-NF-κB (1:1,000, ab16502), anti-p-NF-κB (1:1,000, ab86299) and GAPDH (1:1,000, ab8245) all from Abcam.

    Techniques: Mouse Assay, In Vivo, Quantitative RT-PCR, Western Blot, Immunofluorescence

    Working model of salidroside (SR) against skin cancer. SR prevents the carcinogenesis of mouse skin tissue initiated by DMBA/TPA. SR acts as a drug to suppress skin tumors in mice by inactivating the IκBα/NF-κB pathway, thus reducing the secretion of pro-inflammatory cytokines. In addition, the p53 and caspase-3 signaling pathways were enhanced by SR, resulting in apoptosis to prevent skin carcinogenesis. SR, salidroside. DMBA, 7,12-dimethylbenz( a )anthracene. TPA, 12- O -tetradecanoylphorbol-13-acetate.

    Journal: Oncology Reports

    Article Title: Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a mouse model through suppression of inflammation and promotion of apoptosis

    doi: 10.3892/or.2018.6381

    Figure Lengend Snippet: Working model of salidroside (SR) against skin cancer. SR prevents the carcinogenesis of mouse skin tissue initiated by DMBA/TPA. SR acts as a drug to suppress skin tumors in mice by inactivating the IκBα/NF-κB pathway, thus reducing the secretion of pro-inflammatory cytokines. In addition, the p53 and caspase-3 signaling pathways were enhanced by SR, resulting in apoptosis to prevent skin carcinogenesis. SR, salidroside. DMBA, 7,12-dimethylbenz( a )anthracene. TPA, 12- O -tetradecanoylphorbol-13-acetate.

    Article Snippet: The primary antibodies used in our study included: anti-p21 (1:1,000, ab86696), anti-PUMA (1:1,000, ab9643), anti-Bax (1:1,000, ab32503), anti-p53 (1:1,000, ab131442), anti-caspase-3 (1:1,000, ab90437), anti-IκBα (1:1,000, ab32518), anti-p-IκBα (1:1,000, ab133462), anti-NF-κB (1:1,000, ab16502), anti-p-NF-κB (1:1,000, ab86299) and GAPDH (1:1,000, ab8245) all from Abcam.

    Techniques: Mouse Assay

    SR suppresses the inflammation response in DMBA-induced cells in vitro . (A) Normal human epidermal keratinocytes, HaCaT, human hypertrophic scar fibroblasts (HSFs) and human normal liver cell line L02 were treated with different concnetrations of SR (0, 5, 10, 20, 40, 80 and 160 µM) for 48 h. Then, all cells were harvested for MTT assay. Normal human epidermal keratinocytes, HaCaT, were treated with 20 µg/ml DMBA for 48 h with or without SR treatment at 40, 80 and 160 µM. Then, all cells were harvested for the following research. (B) Western blot analysis was used to determine levels in phosphorylated (p)-IκBα, IκBα and p-NF-κB in cells treated under various conditions. (C) Cytokines of TNF-α, IL-1β, IL-18, IL-6, COX2 and TGF-β1 were evaluated by RT-qPCR analysis. (D) The immunofluorescence analysis was used to assess NF-κB phosphorylation. Data are presented as mean ± SEM (n=10). + P

    Journal: Oncology Reports

    Article Title: Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a mouse model through suppression of inflammation and promotion of apoptosis

    doi: 10.3892/or.2018.6381

    Figure Lengend Snippet: SR suppresses the inflammation response in DMBA-induced cells in vitro . (A) Normal human epidermal keratinocytes, HaCaT, human hypertrophic scar fibroblasts (HSFs) and human normal liver cell line L02 were treated with different concnetrations of SR (0, 5, 10, 20, 40, 80 and 160 µM) for 48 h. Then, all cells were harvested for MTT assay. Normal human epidermal keratinocytes, HaCaT, were treated with 20 µg/ml DMBA for 48 h with or without SR treatment at 40, 80 and 160 µM. Then, all cells were harvested for the following research. (B) Western blot analysis was used to determine levels in phosphorylated (p)-IκBα, IκBα and p-NF-κB in cells treated under various conditions. (C) Cytokines of TNF-α, IL-1β, IL-18, IL-6, COX2 and TGF-β1 were evaluated by RT-qPCR analysis. (D) The immunofluorescence analysis was used to assess NF-κB phosphorylation. Data are presented as mean ± SEM (n=10). + P

    Article Snippet: The primary antibodies used in our study included: anti-p21 (1:1,000, ab86696), anti-PUMA (1:1,000, ab9643), anti-Bax (1:1,000, ab32503), anti-p53 (1:1,000, ab131442), anti-caspase-3 (1:1,000, ab90437), anti-IκBα (1:1,000, ab32518), anti-p-IκBα (1:1,000, ab133462), anti-NF-κB (1:1,000, ab16502), anti-p-NF-κB (1:1,000, ab86299) and GAPDH (1:1,000, ab8245) all from Abcam.

    Techniques: In Vitro, MTT Assay, Western Blot, Quantitative RT-PCR, Immunofluorescence

    SR-mediated suppression of inflammation response in cells is time-dependent. Normal human epidermal keratinocytes (HaCaT) were pre-treated with 80 µM SR for the indicated time. Then, all cells were exposed to TNF-α at 50 ng/ml for 1 h. (A) The representative images of protein bands of phosphorylated(p)-IκBα, IκBα and p-NF-κB in cells. The quantification of (B) p-IκBα, (C) IκBα and (D) p-NF-κB is displayed. Data are presented as mean ± SEM (n=10). + P

    Journal: Oncology Reports

    Article Title: Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a mouse model through suppression of inflammation and promotion of apoptosis

    doi: 10.3892/or.2018.6381

    Figure Lengend Snippet: SR-mediated suppression of inflammation response in cells is time-dependent. Normal human epidermal keratinocytes (HaCaT) were pre-treated with 80 µM SR for the indicated time. Then, all cells were exposed to TNF-α at 50 ng/ml for 1 h. (A) The representative images of protein bands of phosphorylated(p)-IκBα, IκBα and p-NF-κB in cells. The quantification of (B) p-IκBα, (C) IκBα and (D) p-NF-κB is displayed. Data are presented as mean ± SEM (n=10). + P

    Article Snippet: The primary antibodies used in our study included: anti-p21 (1:1,000, ab86696), anti-PUMA (1:1,000, ab9643), anti-Bax (1:1,000, ab32503), anti-p53 (1:1,000, ab131442), anti-caspase-3 (1:1,000, ab90437), anti-IκBα (1:1,000, ab32518), anti-p-IκBα (1:1,000, ab133462), anti-NF-κB (1:1,000, ab16502), anti-p-NF-κB (1:1,000, ab86299) and GAPDH (1:1,000, ab8245) all from Abcam.

    Techniques:

    Immunofluorescence microscopy and Western blot of IκBα in PI-resistant MM cell lines A/B. Immunofluorescence microscopy, U266PSR (A) and 8226B25 (B) PI-resistant cells showed an increase in IκBα (red) after treatment with selinexor/BTZ compared with untreated control or single-agent BTZ or selinexor. C/D. Selinexor/BTZ combination treatment increased IκBα protein in U266PSR (331%) and 8226B25 (312%) cells compared with untreated control or single-agent BTZ or selinexor (n=4).

    Journal: Oncotarget

    Article Title: XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma

    doi: 10.18632/oncotarget.12969

    Figure Lengend Snippet: Immunofluorescence microscopy and Western blot of IκBα in PI-resistant MM cell lines A/B. Immunofluorescence microscopy, U266PSR (A) and 8226B25 (B) PI-resistant cells showed an increase in IκBα (red) after treatment with selinexor/BTZ compared with untreated control or single-agent BTZ or selinexor. C/D. Selinexor/BTZ combination treatment increased IκBα protein in U266PSR (331%) and 8226B25 (312%) cells compared with untreated control or single-agent BTZ or selinexor (n=4).

    Article Snippet: Cells were incubated with primary antibodies to IκBα (E130/Abcam) and NFκB (L8F6/Cell Signaling).

    Techniques: Immunofluorescence, Microscopy, Western Blot

    BTZ/selinexor synergy may be linked to IκBα expression and downregulation of NFκB transcriptional activity A. IM-9 and 8226 MM cells were transfected with 40 nM siRNA against IκBα or control siRNA. 24 hours posttransfection, the cells were treated with selinexor for 72 hours and IC 50 determined. IκBα knockdown reduced toxicity to KPT-330 compared with control siRNA in both IM-9 ( P = 0.023) and 8226 ( P = 0.0062) cells. B. 8226, 8226B25, and H929 human MM cells were transfected with 40 nM IκBα siRNA. 48 hours after transfection, scram siRNA control and IκBα siRNA knockdown cells were treated with 100 nM selinexor ± BTZ. IκBα knockdown reduced apoptosis (activated caspase 3) in selinexor/BTZ-treated cells ( P = 0.0086) compared with control siRNA. C. Chemiluminescent transcription factor assay. MM.1S cells were pretreated with 1 μM selinexor ± 100 nM BTZ for 2 hours and then exposed to 20 ng/mL of TNFα for 4 hours in serum-free media. TNFα exposure induced NFκB transcriptional activity 6-fold. Single-agent selinexor ( P = 0.000023) and BTZ ( P = 0.00024) lowered NFκB transcriptional activity, and the combination of selinexor and BTZ further reduced the activity to 3-fold below baseline ( P = 0.00013) (no TNFα). (Inset) Western blots of protein from 8226 selinexor/bortezomib-treated cells showed a decrease in NFκB-mediated IAP-1 (84%), IAP-2 (72.8%), c-Myc (62%), and cyclin D2 (42%) protein expression compared with untreated controls. Protein loading was confirmed by GAPDH expression.

    Journal: Oncotarget

    Article Title: XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma

    doi: 10.18632/oncotarget.12969

    Figure Lengend Snippet: BTZ/selinexor synergy may be linked to IκBα expression and downregulation of NFκB transcriptional activity A. IM-9 and 8226 MM cells were transfected with 40 nM siRNA against IκBα or control siRNA. 24 hours posttransfection, the cells were treated with selinexor for 72 hours and IC 50 determined. IκBα knockdown reduced toxicity to KPT-330 compared with control siRNA in both IM-9 ( P = 0.023) and 8226 ( P = 0.0062) cells. B. 8226, 8226B25, and H929 human MM cells were transfected with 40 nM IκBα siRNA. 48 hours after transfection, scram siRNA control and IκBα siRNA knockdown cells were treated with 100 nM selinexor ± BTZ. IκBα knockdown reduced apoptosis (activated caspase 3) in selinexor/BTZ-treated cells ( P = 0.0086) compared with control siRNA. C. Chemiluminescent transcription factor assay. MM.1S cells were pretreated with 1 μM selinexor ± 100 nM BTZ for 2 hours and then exposed to 20 ng/mL of TNFα for 4 hours in serum-free media. TNFα exposure induced NFκB transcriptional activity 6-fold. Single-agent selinexor ( P = 0.000023) and BTZ ( P = 0.00024) lowered NFκB transcriptional activity, and the combination of selinexor and BTZ further reduced the activity to 3-fold below baseline ( P = 0.00013) (no TNFα). (Inset) Western blots of protein from 8226 selinexor/bortezomib-treated cells showed a decrease in NFκB-mediated IAP-1 (84%), IAP-2 (72.8%), c-Myc (62%), and cyclin D2 (42%) protein expression compared with untreated controls. Protein loading was confirmed by GAPDH expression.

    Article Snippet: Cells were incubated with primary antibodies to IκBα (E130/Abcam) and NFκB (L8F6/Cell Signaling).

    Techniques: Expressing, Activity Assay, Transfection, Transcription Factor Assay, Western Blot

    ImageStream flow cytometry A. 8226B25 human MM cells were treated for 20 hours with BTZ, SEL, or SEL/BTZ. An Imagestream histogram was generated using the similarity feature showing co-localization of IκBα (green) to the nuclear stain, DAPI (purple). IκBα shifted from the cytoplasm to the nucleus in BTZ-treated cells ( P = 0.006), further shifted in SEL-treated cells compared with non-treated controls ( P = 0.000067). However, the largest nuclear shift was seen in the SEL/BTZ combination treatment (n = 3) ( P = 0.000057). B. Images of representative cells in real time, visually showing increased IκBα protein in the cell nuclei. Far right column shows the IκBα nuclear/cytoplasmic ratio for 5000 cells from each treatment group (n =3). *The similarity feature is the log-transformed Pearson correlation coefficient and is a measure of the degree to which two images are linearly correlated within a masked region.

    Journal: Oncotarget

    Article Title: XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma

    doi: 10.18632/oncotarget.12969

    Figure Lengend Snippet: ImageStream flow cytometry A. 8226B25 human MM cells were treated for 20 hours with BTZ, SEL, or SEL/BTZ. An Imagestream histogram was generated using the similarity feature showing co-localization of IκBα (green) to the nuclear stain, DAPI (purple). IκBα shifted from the cytoplasm to the nucleus in BTZ-treated cells ( P = 0.006), further shifted in SEL-treated cells compared with non-treated controls ( P = 0.000067). However, the largest nuclear shift was seen in the SEL/BTZ combination treatment (n = 3) ( P = 0.000057). B. Images of representative cells in real time, visually showing increased IκBα protein in the cell nuclei. Far right column shows the IκBα nuclear/cytoplasmic ratio for 5000 cells from each treatment group (n =3). *The similarity feature is the log-transformed Pearson correlation coefficient and is a measure of the degree to which two images are linearly correlated within a masked region.

    Article Snippet: Cells were incubated with primary antibodies to IκBα (E130/Abcam) and NFκB (L8F6/Cell Signaling).

    Techniques: Flow Cytometry, Cytometry, Generated, Staining, Transformation Assay

    Selinexor promotes NFκB-IκBα binding A. Proximity ligation assay for 8226B25 PI-resistant MM cells (3×10 6 /ml) treated and stained with antibodies for NFκB and IκBα. Selinexor (KPT-330) in combination with BTZ increased proximity co-localization of NFκB and IκBα up to 12-fold over untreated and single-agent BTZ or selinexor. Green fluorescence denotes the cytoplasm, and blue indicates the nucleus (DAPI). B. Selinexor/BTZ significantly increased the number of NFκB-IκBα foci in the nucleus versus no drug or single-agent selinexor or BTZ ( P ≤ 0.00077) (n=3, 50 cells per assay). Inset: Selinexor treatment did not affect XPO1 protein expression at 4 hours as shown by Western blot.

    Journal: Oncotarget

    Article Title: XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma

    doi: 10.18632/oncotarget.12969

    Figure Lengend Snippet: Selinexor promotes NFκB-IκBα binding A. Proximity ligation assay for 8226B25 PI-resistant MM cells (3×10 6 /ml) treated and stained with antibodies for NFκB and IκBα. Selinexor (KPT-330) in combination with BTZ increased proximity co-localization of NFκB and IκBα up to 12-fold over untreated and single-agent BTZ or selinexor. Green fluorescence denotes the cytoplasm, and blue indicates the nucleus (DAPI). B. Selinexor/BTZ significantly increased the number of NFκB-IκBα foci in the nucleus versus no drug or single-agent selinexor or BTZ ( P ≤ 0.00077) (n=3, 50 cells per assay). Inset: Selinexor treatment did not affect XPO1 protein expression at 4 hours as shown by Western blot.

    Article Snippet: Cells were incubated with primary antibodies to IκBα (E130/Abcam) and NFκB (L8F6/Cell Signaling).

    Techniques: Binding Assay, Proximity Ligation Assay, Staining, Fluorescence, Expressing, Western Blot

    Immunofluorescence microscopy of IκBα in patient MM treated with selinexor Bone marrow aspirates from newly diagnosed, relapsed, and refractory MM patients were treated ex vivo with 100-300 nM selinexor for 20 hours. MM cells were identified by kappa/lambda light chain antigen staining (green), nuclei identified by DAPI (blue) staining, and IκBα protein (red). All patient MM samples showed an increase in IκBα protein, especially in the cell nuclei with ex vivo selinexor treatment compared with untreated controls from the same patient.

    Journal: Oncotarget

    Article Title: XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IκBα and overcomes acquired proteasome inhibitor resistance in human multiple myeloma

    doi: 10.18632/oncotarget.12969

    Figure Lengend Snippet: Immunofluorescence microscopy of IκBα in patient MM treated with selinexor Bone marrow aspirates from newly diagnosed, relapsed, and refractory MM patients were treated ex vivo with 100-300 nM selinexor for 20 hours. MM cells were identified by kappa/lambda light chain antigen staining (green), nuclei identified by DAPI (blue) staining, and IκBα protein (red). All patient MM samples showed an increase in IκBα protein, especially in the cell nuclei with ex vivo selinexor treatment compared with untreated controls from the same patient.

    Article Snippet: Cells were incubated with primary antibodies to IκBα (E130/Abcam) and NFκB (L8F6/Cell Signaling).

    Techniques: Immunofluorescence, Microscopy, Ex Vivo, Staining

    Schematic representation of TG2-regulated signaling. Association of TG2 with IκBα results in its rapid degradation via non-proteasomal pathway and liberates the p65/RelA:p50 NF-κB complex. TG2 also associates in complex with the p65/RealA subunit of NF-κB and translocates to the nucleus where it binds to the cognate NF-κB binding site on the HIF-1α promoter and results in its transcription regulation. Increased expression of HIF-1α under normoxic conditions results in increased expression of various downstream target genes such as transcription repressors Zeb1, Zeb2, Snail1 and Twist , which induce EMT and promote cell survival, invasiveness, and cellular plasticity. Overall, these TG2-regulated changes contribute to increased chemoresistance and metastatic potential.

    Journal: PLoS ONE

    Article Title: Tissue Transglutaminase Constitutively Activates HIF-1? Promoter and Nuclear Factor-?B via a Non-Canonical Pathway

    doi: 10.1371/journal.pone.0049321

    Figure Lengend Snippet: Schematic representation of TG2-regulated signaling. Association of TG2 with IκBα results in its rapid degradation via non-proteasomal pathway and liberates the p65/RelA:p50 NF-κB complex. TG2 also associates in complex with the p65/RealA subunit of NF-κB and translocates to the nucleus where it binds to the cognate NF-κB binding site on the HIF-1α promoter and results in its transcription regulation. Increased expression of HIF-1α under normoxic conditions results in increased expression of various downstream target genes such as transcription repressors Zeb1, Zeb2, Snail1 and Twist , which induce EMT and promote cell survival, invasiveness, and cellular plasticity. Overall, these TG2-regulated changes contribute to increased chemoresistance and metastatic potential.

    Article Snippet: Anti-TG2, anti-Snail, and anti-β-actin antibodies were purchased from Abcam; anti-IκBα antibodies were purchased from Imgenex Corporation; and anti-p65/RelA, anti-ZEB1, and anti-Twist antibodies were purchased from Santa Cruz Biotechnology.

    Techniques: Binding Assay, Expressing

    TG2 expression correlates with constitutive NF-κB activation. A- Western blot analyses of nuclear and cytosolic fractions prepared from vector, TG2-WT, and TG2-C277S-expressing MCF10A cells. Membranes were probed with either anti-TG2 or anti-p65/RelA antibodies, stripped, and reprobed with anti-IκBα, GAPDH or anti-histone antibodies to determine even protein loading and purity of cytosolic and nuclear fractions, respectively. The low IκBα protein levels in cytosolic fractions were related to TG2 expression, as suggested by the even β actin band in all the three cytosolic fractions (data not shown). B- Detection of p65/RelA in the nuclear fraction of TG2-C277S transfected MCF10A cells and C- in MCF-7/RT cells after transfection with either the control or TG2-specific shRNA. Membranes were reprobed with anti-IκBα, GAPDH or anti-histone antibodies to ensure the even loading and purity of cytosolic and nuclear fractions. D- Quantitative RT-PCR array showing relative changes in the expression of NF-κB target genes in TG2-C277S-transfected MCF10A cells and MCF10A-vector cells after stimulation with TNFα (10 ng/mL) for the indicated times. The Y-axis denotes the fold-expression, and the x-axis denotes the genes. Expression of GAPDH, β actin, and 18S ribosomal RNA was used to normalize the variable template loading. E- RT-PCR analysis for basal and TNFα-induced expression of selected NF-κB target genes in TG2-C277S and control vector-transfected MCF10A cells. F- Immunoblot analysis for basal expression of indicated NF-κB target genes in TG2-C277S and vector-transfected MCF10A cells. Results shown are from a representative experiment repeated at least 2 or 3 times with similar results.

    Journal: PLoS ONE

    Article Title: Tissue Transglutaminase Constitutively Activates HIF-1? Promoter and Nuclear Factor-?B via a Non-Canonical Pathway

    doi: 10.1371/journal.pone.0049321

    Figure Lengend Snippet: TG2 expression correlates with constitutive NF-κB activation. A- Western blot analyses of nuclear and cytosolic fractions prepared from vector, TG2-WT, and TG2-C277S-expressing MCF10A cells. Membranes were probed with either anti-TG2 or anti-p65/RelA antibodies, stripped, and reprobed with anti-IκBα, GAPDH or anti-histone antibodies to determine even protein loading and purity of cytosolic and nuclear fractions, respectively. The low IκBα protein levels in cytosolic fractions were related to TG2 expression, as suggested by the even β actin band in all the three cytosolic fractions (data not shown). B- Detection of p65/RelA in the nuclear fraction of TG2-C277S transfected MCF10A cells and C- in MCF-7/RT cells after transfection with either the control or TG2-specific shRNA. Membranes were reprobed with anti-IκBα, GAPDH or anti-histone antibodies to ensure the even loading and purity of cytosolic and nuclear fractions. D- Quantitative RT-PCR array showing relative changes in the expression of NF-κB target genes in TG2-C277S-transfected MCF10A cells and MCF10A-vector cells after stimulation with TNFα (10 ng/mL) for the indicated times. The Y-axis denotes the fold-expression, and the x-axis denotes the genes. Expression of GAPDH, β actin, and 18S ribosomal RNA was used to normalize the variable template loading. E- RT-PCR analysis for basal and TNFα-induced expression of selected NF-κB target genes in TG2-C277S and control vector-transfected MCF10A cells. F- Immunoblot analysis for basal expression of indicated NF-κB target genes in TG2-C277S and vector-transfected MCF10A cells. Results shown are from a representative experiment repeated at least 2 or 3 times with similar results.

    Article Snippet: Anti-TG2, anti-Snail, and anti-β-actin antibodies were purchased from Abcam; anti-IκBα antibodies were purchased from Imgenex Corporation; and anti-p65/RelA, anti-ZEB1, and anti-Twist antibodies were purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, Activation Assay, Western Blot, Plasmid Preparation, Transfection, shRNA, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    TG2 mediate IκBα degradation via non-proteasomal pathway and results in HIF-1α expression. A- Total cell extracts prepared from MCF10A-Vec, MCF10A TG2-C277S, drug-sensitive (MCF-7/WT) and drug resistant (MCF-7/RT) MCF-7 cell were immunoprecipitated with anti-p65/RelA or anti-IκBα antibody and immunoblotted with anti-TG2, anti- p65/RelA, or anti- IκBα antibody. Total cell extracts (15 µg protein) were also directly subjected to immunoblotting to determine TG2 and p65/RelA levels. B- MCF10A-Vec and MCF10A TG2-C277S cells were incubated with medium alone (Cont.) or medium containing the indicated proteasomal inhibitor [Bortezomib (20 nM), N-acetyl-leucyl-leucyl-norleucinal (ALLN) (50 µg/mL), and MG-132 (1 µM)] for 2 hours. At the end of incubation, cells extracts were prepared and subjected to immunoblot analysis, using anti-TG2, anti-IκBα and anti-p21 antibody (p21 served as a positive control) and β-actin to ensure equal protein loading. C- RT-PCR analysis showing the basal transcript level of HIF-1α in MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT and MCF7/RT cells. Results shown are from a representative experiment repeated at least 2 times with similar results. D- Immunoblot analysis showing the basal HIF-1α and TG2 protein expression in MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT and MCF7/RT cells under normoxic conditions.

    Journal: PLoS ONE

    Article Title: Tissue Transglutaminase Constitutively Activates HIF-1? Promoter and Nuclear Factor-?B via a Non-Canonical Pathway

    doi: 10.1371/journal.pone.0049321

    Figure Lengend Snippet: TG2 mediate IκBα degradation via non-proteasomal pathway and results in HIF-1α expression. A- Total cell extracts prepared from MCF10A-Vec, MCF10A TG2-C277S, drug-sensitive (MCF-7/WT) and drug resistant (MCF-7/RT) MCF-7 cell were immunoprecipitated with anti-p65/RelA or anti-IκBα antibody and immunoblotted with anti-TG2, anti- p65/RelA, or anti- IκBα antibody. Total cell extracts (15 µg protein) were also directly subjected to immunoblotting to determine TG2 and p65/RelA levels. B- MCF10A-Vec and MCF10A TG2-C277S cells were incubated with medium alone (Cont.) or medium containing the indicated proteasomal inhibitor [Bortezomib (20 nM), N-acetyl-leucyl-leucyl-norleucinal (ALLN) (50 µg/mL), and MG-132 (1 µM)] for 2 hours. At the end of incubation, cells extracts were prepared and subjected to immunoblot analysis, using anti-TG2, anti-IκBα and anti-p21 antibody (p21 served as a positive control) and β-actin to ensure equal protein loading. C- RT-PCR analysis showing the basal transcript level of HIF-1α in MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT and MCF7/RT cells. Results shown are from a representative experiment repeated at least 2 times with similar results. D- Immunoblot analysis showing the basal HIF-1α and TG2 protein expression in MCF10A-Vec, MCF10A TG2-C277S, MCF7-WT and MCF7/RT cells under normoxic conditions.

    Article Snippet: Anti-TG2, anti-Snail, and anti-β-actin antibodies were purchased from Abcam; anti-IκBα antibodies were purchased from Imgenex Corporation; and anti-p65/RelA, anti-ZEB1, and anti-Twist antibodies were purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, Immunoprecipitation, Incubation, Positive Control, Reverse Transcription Polymerase Chain Reaction

    Doxorubicin-induced NF-κB activation enhances DNA damage and apoptosis in A172 glioblastoma cells. (A), Ectopic expression of IκBα-SR. A172 glioblastoma cells were stably transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B), Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of A172 cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C), Inhibition of NF-κB transcriptional activity by IκBα-SR. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα or for 24 hrs with 0.8 μg/ml Doxorubicin and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D), Enhancement of TNFα-induced apoptosis by NF-κB inhibition. A172 cells transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. (E), NF-κB promotes Doxorubicin-induced DNA damage. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (F), NF-κB promotes Doxorubicin-induced apoptosis. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium-iodide stained nuclei. Median (E) or mean (C, D and F) + S.D. of three independent experiments are shown; * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

    doi: 10.1111/j.1582-4934.2009.00888.x

    Figure Lengend Snippet: Doxorubicin-induced NF-κB activation enhances DNA damage and apoptosis in A172 glioblastoma cells. (A), Ectopic expression of IκBα-SR. A172 glioblastoma cells were stably transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B), Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of A172 cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C), Inhibition of NF-κB transcriptional activity by IκBα-SR. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα or for 24 hrs with 0.8 μg/ml Doxorubicin and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D), Enhancement of TNFα-induced apoptosis by NF-κB inhibition. A172 cells transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. (E), NF-κB promotes Doxorubicin-induced DNA damage. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (F), NF-κB promotes Doxorubicin-induced apoptosis. A172 cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium-iodide stained nuclei. Median (E) or mean (C, D and F) + S.D. of three independent experiments are shown; * P

    Article Snippet: Western blot analysis Western blotting was performed as previously described [ ] using the following antibodies: mouse anti-β-actin (Sigma), mouse anti-Bcl2 (BD Pharmingen), mouse anti-Bcl-XL (BD Biosciences, San Jose, CA, USA), goat anti-cIAP1 (R & D Systems, Inc.), rabbit anti-cIAP2 (Epitomics), rabbit anti-IκBα (Santa Cruz Biotechnology Inc., Heidelberg, Germany), mouse anti-p53 (BD Biosciences) and mouse anti-XIAP (BD Biosciences).

    Techniques: Activation Assay, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Western Blot, Inhibition, Binding Assay, Activity Assay, Transfection, Luciferase, Construct, FACS, Staining, Single Cell Gel Electrophoresis

    Effect of NF-κB inhibition on Doxorubicin-induced cellular stress response. (A) Effect of NF-κB inhibition on Doxorubicin uptake and efflux. U87MG cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, Doxorubicin content was assessed by flow cytometry. Mean + S.D. of three independent experiments performed in triplicate are shown. (B) Effect of NF-κB inhibition on Doxorubicin-induced cell cycle arrest. U87MG cells stably transduced with control vector or a vector containing IκBα-SR were treated with 0.8 μg/ml Doxorubicin for 18 hrs. followed by a complete exchange of medium. After the indicated timepoints, cell cycle progression was assessed by flow cytometry. Mean of three independent experiments performed in triplicate are shown.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

    doi: 10.1111/j.1582-4934.2009.00888.x

    Figure Lengend Snippet: Effect of NF-κB inhibition on Doxorubicin-induced cellular stress response. (A) Effect of NF-κB inhibition on Doxorubicin uptake and efflux. U87MG cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, Doxorubicin content was assessed by flow cytometry. Mean + S.D. of three independent experiments performed in triplicate are shown. (B) Effect of NF-κB inhibition on Doxorubicin-induced cell cycle arrest. U87MG cells stably transduced with control vector or a vector containing IκBα-SR were treated with 0.8 μg/ml Doxorubicin for 18 hrs. followed by a complete exchange of medium. After the indicated timepoints, cell cycle progression was assessed by flow cytometry. Mean of three independent experiments performed in triplicate are shown.

    Article Snippet: Western blot analysis Western blotting was performed as previously described [ ] using the following antibodies: mouse anti-β-actin (Sigma), mouse anti-Bcl2 (BD Pharmingen), mouse anti-Bcl-XL (BD Biosciences, San Jose, CA, USA), goat anti-cIAP1 (R & D Systems, Inc.), rabbit anti-cIAP2 (Epitomics), rabbit anti-IκBα (Santa Cruz Biotechnology Inc., Heidelberg, Germany), mouse anti-p53 (BD Biosciences) and mouse anti-XIAP (BD Biosciences).

    Techniques: Inhibition, Stable Transfection, Transduction, Plasmid Preparation, Flow Cytometry, Cytometry

    NF-κB enhances DNA intercalator-induced DNA damage. (A) U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were continuously treated for indicated times with 0.8 μg/ml Doxorubicin (U87MG) or 1 μg/ml Doxorubicin (T98G), 2 μM Daunorubicin or 2.5 μM Mitoxantrone and DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (B) U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with Doxorubicin (U87MG: 0.8 μg/ml for 18 hrs; T98G: 1 μg/ml for 18 hrs), 2 μM Daunorubicin for 18 hrs or 2.5 μM Mitoxantrone for 3 hrs (U87MG) or 6 hrs (T98G), followed by a complete exchange of medium. After the indicated time points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. In (C) and (D), U87MG cells stably transduced with control vector were treated with 0.8 μg/ml Doxorubicin in the absence (white bars) or presence (hatched bars) of 10 μg/ml CHX for 18 hrs, followed by a complete exchange of medium and re-addition of CHX. DNA damage was assayed after 6 and 24 hrs by Comet assay and is displayed as Olive Tail Moment (C), apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei (D). In (E) and (F), U87MG cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated for 18 hrs with 0.8 μg/ml Doxorubicin in the absence or presence of 50 μM zVAD.fmk, followed by a complete exchange of medium and re-addition of zVAD.fmk. DNA damage was assayed after 24 hrs by Comet assay and is displayed as Olive Tail Moment (E), apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei (D). Median (A–C, E) or mean (D, F) + S.D. of three independent experiments are shown; * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

    doi: 10.1111/j.1582-4934.2009.00888.x

    Figure Lengend Snippet: NF-κB enhances DNA intercalator-induced DNA damage. (A) U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were continuously treated for indicated times with 0.8 μg/ml Doxorubicin (U87MG) or 1 μg/ml Doxorubicin (T98G), 2 μM Daunorubicin or 2.5 μM Mitoxantrone and DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. (B) U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with Doxorubicin (U87MG: 0.8 μg/ml for 18 hrs; T98G: 1 μg/ml for 18 hrs), 2 μM Daunorubicin for 18 hrs or 2.5 μM Mitoxantrone for 3 hrs (U87MG) or 6 hrs (T98G), followed by a complete exchange of medium. After the indicated time points, DNA damage was assayed by Comet assay and is displayed as Olive Tail Moment. In (C) and (D), U87MG cells stably transduced with control vector were treated with 0.8 μg/ml Doxorubicin in the absence (white bars) or presence (hatched bars) of 10 μg/ml CHX for 18 hrs, followed by a complete exchange of medium and re-addition of CHX. DNA damage was assayed after 6 and 24 hrs by Comet assay and is displayed as Olive Tail Moment (C), apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei (D). In (E) and (F), U87MG cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated for 18 hrs with 0.8 μg/ml Doxorubicin in the absence or presence of 50 μM zVAD.fmk, followed by a complete exchange of medium and re-addition of zVAD.fmk. DNA damage was assayed after 24 hrs by Comet assay and is displayed as Olive Tail Moment (E), apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei (D). Median (A–C, E) or mean (D, F) + S.D. of three independent experiments are shown; * P

    Article Snippet: Western blot analysis Western blotting was performed as previously described [ ] using the following antibodies: mouse anti-β-actin (Sigma), mouse anti-Bcl2 (BD Pharmingen), mouse anti-Bcl-XL (BD Biosciences, San Jose, CA, USA), goat anti-cIAP1 (R & D Systems, Inc.), rabbit anti-cIAP2 (Epitomics), rabbit anti-IκBα (Santa Cruz Biotechnology Inc., Heidelberg, Germany), mouse anti-p53 (BD Biosciences) and mouse anti-XIAP (BD Biosciences).

    Techniques: Stable Transfection, Transduction, Plasmid Preparation, Single Cell Gel Electrophoresis, FACS, Staining

    NF-κB promotes DNA intercalator-induced apoptosis. In (A), U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin for 18 hrs, 2 μM Daunorubicin for 18 hrs or 2.5 μM Mitoxantrone for 3 hrs (U87MG) or 6 hrs (T98G), followed by a complete exchange of medium. After the indicated time points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. In (B), U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were continuously treated for indicated times with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin, 2 μM Daunorubicin or 2.5 μM Mitoxantrone and apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei. In (C) and (D), U87MG cells were transiently transfected twice consecutively with p65 siRNA or control siRNA. Forty-eight hours after the second transfection cells were reseeded (seed) and the next day treated with 0.8 μg/ml Doxorubicin for 18 hrs (treat), followed by a complete exchange of medium. In (C), Protein expression of p65 was analysed by Western blotting the day of seeding, treating and 0 hr, 24 hrs and 48 hrs after drug removal. β-actin served as loading control. In (D), U87MG cells were transiently transfected twice with control siRNA (white bars) or p65 siRNA (hatched bars). After the indicated time points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. In (E), U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated for 24 hrs with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin, 2 μM Daunorubicin or 2.5 μM Mitoxantrone and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. In (F), U87MG cells stably transduced with control vector or a vector containing IκBα-SR were treated for 12 and 18 hrs with 0.8 μg/ml Doxorubicin and expression levels of anti-apoptotic proteins were determined by Western blotting. EBV-transformed B cells were used as positive control (PC) for cIAP2 expression. Actin served as loading control in (A), (B), (D) and (E), mean + S.D. of three independent experiments performed in triplicates is shown. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

    doi: 10.1111/j.1582-4934.2009.00888.x

    Figure Lengend Snippet: NF-κB promotes DNA intercalator-induced apoptosis. In (A), U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin for 18 hrs, 2 μM Daunorubicin for 18 hrs or 2.5 μM Mitoxantrone for 3 hrs (U87MG) or 6 hrs (T98G), followed by a complete exchange of medium. After the indicated time points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. In (B), U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were continuously treated for indicated times with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin, 2 μM Daunorubicin or 2.5 μM Mitoxantrone and apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei. In (C) and (D), U87MG cells were transiently transfected twice consecutively with p65 siRNA or control siRNA. Forty-eight hours after the second transfection cells were reseeded (seed) and the next day treated with 0.8 μg/ml Doxorubicin for 18 hrs (treat), followed by a complete exchange of medium. In (C), Protein expression of p65 was analysed by Western blotting the day of seeding, treating and 0 hr, 24 hrs and 48 hrs after drug removal. β-actin served as loading control. In (D), U87MG cells were transiently transfected twice with control siRNA (white bars) or p65 siRNA (hatched bars). After the indicated time points, apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. In (E), U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were treated for 24 hrs with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin, 2 μM Daunorubicin or 2.5 μM Mitoxantrone and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. In (F), U87MG cells stably transduced with control vector or a vector containing IκBα-SR were treated for 12 and 18 hrs with 0.8 μg/ml Doxorubicin and expression levels of anti-apoptotic proteins were determined by Western blotting. EBV-transformed B cells were used as positive control (PC) for cIAP2 expression. Actin served as loading control in (A), (B), (D) and (E), mean + S.D. of three independent experiments performed in triplicates is shown. * P

    Article Snippet: Western blot analysis Western blotting was performed as previously described [ ] using the following antibodies: mouse anti-β-actin (Sigma), mouse anti-Bcl2 (BD Pharmingen), mouse anti-Bcl-XL (BD Biosciences, San Jose, CA, USA), goat anti-cIAP1 (R & D Systems, Inc.), rabbit anti-cIAP2 (Epitomics), rabbit anti-IκBα (Santa Cruz Biotechnology Inc., Heidelberg, Germany), mouse anti-p53 (BD Biosciences) and mouse anti-XIAP (BD Biosciences).

    Techniques: Stable Transfection, Transduction, Plasmid Preparation, FACS, Staining, Transfection, Expressing, Western Blot, Luciferase, Activity Assay, Transformation Assay, Positive Control

    NF-κB enhances Doxorubicin-induced apoptosis in a p53-independent manner. (A) Knockdown of p53. U87MG cells stably transduced with control vector or a vector containing IκBα-SR and stably infected with empty vector (EV), control shRNA or p53 shRNA vector were treated with 0.8 μg/ml Doxorubicin for 18 hrs. Protein expression of p53 was analysed by Western blotting. β-actin served as loading control. (B) Effect of p53 knockdown on Doxorubicin-induced apoptosis. U87MG cells stably transduced with control vector (bright bars) or a vector containing IκBα-SR (dark bars) and stably infected with empty vector (EV), control shRNA or p53 shRNA vector were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei. Mean + S.D. of three independent experiments are shown. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

    doi: 10.1111/j.1582-4934.2009.00888.x

    Figure Lengend Snippet: NF-κB enhances Doxorubicin-induced apoptosis in a p53-independent manner. (A) Knockdown of p53. U87MG cells stably transduced with control vector or a vector containing IκBα-SR and stably infected with empty vector (EV), control shRNA or p53 shRNA vector were treated with 0.8 μg/ml Doxorubicin for 18 hrs. Protein expression of p53 was analysed by Western blotting. β-actin served as loading control. (B) Effect of p53 knockdown on Doxorubicin-induced apoptosis. U87MG cells stably transduced with control vector (bright bars) or a vector containing IκBα-SR (dark bars) and stably infected with empty vector (EV), control shRNA or p53 shRNA vector were treated with 0.8 μg/ml Doxorubicin for 18 hrs, followed by a complete exchange of medium. After the indicated time-points, apoptosis was assessed by FACS analysis of DNA fragmentation of propidium iodide stained nuclei. Mean + S.D. of three independent experiments are shown. * P

    Article Snippet: Western blot analysis Western blotting was performed as previously described [ ] using the following antibodies: mouse anti-β-actin (Sigma), mouse anti-Bcl2 (BD Pharmingen), mouse anti-Bcl-XL (BD Biosciences, San Jose, CA, USA), goat anti-cIAP1 (R & D Systems, Inc.), rabbit anti-cIAP2 (Epitomics), rabbit anti-IκBα (Santa Cruz Biotechnology Inc., Heidelberg, Germany), mouse anti-p53 (BD Biosciences) and mouse anti-XIAP (BD Biosciences).

    Techniques: Stable Transfection, Transduction, Plasmid Preparation, Infection, shRNA, Expressing, Western Blot, FACS, Staining

    Generation of glioblastoma cell lines with stable NF-κB inhibition. (A) Ectopic expression of IκBα-SR. U87MG and T98G glioblastoma cells were transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B) Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C) Inhibition of NF-κB transcriptional activity by IκBα-SR. U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D) Enhancement of TNFα-induced apoptosis by NF-κB inhibition. U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. Mean values of three independent triplicate experiments with S.D. are shown; * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Identification of a novel pro-apopotic function of NF-κB in the DNA damage response

    doi: 10.1111/j.1582-4934.2009.00888.x

    Figure Lengend Snippet: Generation of glioblastoma cell lines with stable NF-κB inhibition. (A) Ectopic expression of IκBα-SR. U87MG and T98G glioblastoma cells were transduced with a control vector or a vector containing IκBα-SR. Protein expression of wild-type IκBα and mutant IκBα-SR was determined by Western blot analysis. β-actin served as loading control. (B) Inhibition of NF-κB DNA binding by IκBα-SR. NF-κB DNA binding was assessed by EMSA in nuclear extracts of cells transduced with control vector or a vector containing IκBα-SR that were left untreated or were treated with 0.8 μg/ml (U87MG) or 1 μg/ml (T98G) Doxorubicin for 6 hrs or 10 ng/ml TNFα for 1 hr. (C) Inhibition of NF-κB transcriptional activity by IκBα-SR. U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were transiently transfected with firefly and renilla luciferase gene constructs, treated for 6 hrs with 10 ng/ml TNFα and analysed by dual luciferase assay for induction of NF-κB transcriptional activity. Fold increase in luciferase activity relative to unstimulated control is shown. (D) Enhancement of TNFα-induced apoptosis by NF-κB inhibition. U87MG (left panels) or T98G (right panels) cells stably transduced with control vector (white bars) or a vector containing IκBα-SR (black bars) were left untreated (–TNFα) or were treated with 50 ng/ml TNFα for 48 hrs (+TNFα). Apoptosis was determined by FACS analysis of DNA-fragmentation of propidium iodide stained nuclei. Mean values of three independent triplicate experiments with S.D. are shown; * P

    Article Snippet: Western blot analysis Western blotting was performed as previously described [ ] using the following antibodies: mouse anti-β-actin (Sigma), mouse anti-Bcl2 (BD Pharmingen), mouse anti-Bcl-XL (BD Biosciences, San Jose, CA, USA), goat anti-cIAP1 (R & D Systems, Inc.), rabbit anti-cIAP2 (Epitomics), rabbit anti-IκBα (Santa Cruz Biotechnology Inc., Heidelberg, Germany), mouse anti-p53 (BD Biosciences) and mouse anti-XIAP (BD Biosciences).

    Techniques: Inhibition, Expressing, Transduction, Plasmid Preparation, Mutagenesis, Western Blot, Binding Assay, Activity Assay, Stable Transfection, Transfection, Luciferase, Construct, FACS, Staining

    Effect of β-(1→6)-glucan on phosphorylation of IκB-α, ERK, JNK and p38 in murine macrophage cell line J774a.l. (A) Representative results of membranes being probed for phosphorylated protein, re-probed for total protein, stripped and re-probed for loading control. (B) Fold regulation (ratio phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control ( P ≤ 0.05).

    Journal: Innate immunity

    Article Title: Cellular and molecular mechanisms of fungal β-(l→6)-glucan in macrophages

    doi: 10.1177/1753425915595874

    Figure Lengend Snippet: Effect of β-(1→6)-glucan on phosphorylation of IκB-α, ERK, JNK and p38 in murine macrophage cell line J774a.l. (A) Representative results of membranes being probed for phosphorylated protein, re-probed for total protein, stripped and re-probed for loading control. (B) Fold regulation (ratio phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control ( P ≤ 0.05).

    Article Snippet: The membranes were washed, re-blocked and re-probed overnight with the appropriate Ab: rabbit anti-ERK, rabbit anti-JNK, rabbit anti-p38 and rabbit anti-IκB-α (Santa Cruz Biotechnology, Inc., CA) followed by incubation with peroxidase-conjugated secondary anti-rabbit Abs (Sigma Chemical, MO).

    Techniques:

    Phosphorylation status of IκB-α, ERK and p38 in primary peritoneal macrophages treated with β-(1→6)-glucan. The data are presented as fold regulation (ratio of phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control ( P ≤ 0.05).

    Journal: Innate immunity

    Article Title: Cellular and molecular mechanisms of fungal β-(l→6)-glucan in macrophages

    doi: 10.1177/1753425915595874

    Figure Lengend Snippet: Phosphorylation status of IκB-α, ERK and p38 in primary peritoneal macrophages treated with β-(1→6)-glucan. The data are presented as fold regulation (ratio of phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control ( P ≤ 0.05).

    Article Snippet: The membranes were washed, re-blocked and re-probed overnight with the appropriate Ab: rabbit anti-ERK, rabbit anti-JNK, rabbit anti-p38 and rabbit anti-IκB-α (Santa Cruz Biotechnology, Inc., CA) followed by incubation with peroxidase-conjugated secondary anti-rabbit Abs (Sigma Chemical, MO).

    Techniques:

    DPQ inhibits degradation of IκB-α and subsequent activation of NF-κB in macrophages. Macrophages were stimulated with LPS for indicated time and lysed for protein extraction. The expressions of IκB-α (A) and phosphorylation of NF-κB p65 (B) were detected by Western blot. The experiment was repeated for 3 times with similar results. Upper panel shows the representative immunoblot and lower panel shows the quantitative analysis of the protein expression. Data are expressed as mean ± SEM; n = 3 * P

    Journal: PLoS ONE

    Article Title: PARP-1 Inhibitor, DPQ, Attenuates LPS-Induced Acute Lung Injury through Inhibiting NF-?B-Mediated Inflammatory Response

    doi: 10.1371/journal.pone.0079757

    Figure Lengend Snippet: DPQ inhibits degradation of IκB-α and subsequent activation of NF-κB in macrophages. Macrophages were stimulated with LPS for indicated time and lysed for protein extraction. The expressions of IκB-α (A) and phosphorylation of NF-κB p65 (B) were detected by Western blot. The experiment was repeated for 3 times with similar results. Upper panel shows the representative immunoblot and lower panel shows the quantitative analysis of the protein expression. Data are expressed as mean ± SEM; n = 3 * P

    Article Snippet: Antibodies, Reagents and Mice Rabbit anti-NF-κB-p65 (phospho-p65), rabbit anti-IκB-α, mouse monoclonal anti-GAPDH antibodies were bought from Cell Signaling Technology.

    Techniques: Activation Assay, Protein Extraction, Western Blot, Expressing

    DPQ treatment blocks degradation of IκB-α and subsequent activation of NF-κB. After the mice were treated with LPS for 6 hours, the lung tissues were collected and homogenized for the extraction of total proteins. The expressions of IκB-α (A, C) and phosphorylation of NF-κB p65 (B, D) were detected by Western blot. The experiment was repeated for 3 times with similar results. Lower panel shows the representative immunoblot and upper panel shows the quantitative analysis of the protein expression. Data are expressed as mean ± SEM; n = 3; * P

    Journal: PLoS ONE

    Article Title: PARP-1 Inhibitor, DPQ, Attenuates LPS-Induced Acute Lung Injury through Inhibiting NF-?B-Mediated Inflammatory Response

    doi: 10.1371/journal.pone.0079757

    Figure Lengend Snippet: DPQ treatment blocks degradation of IκB-α and subsequent activation of NF-κB. After the mice were treated with LPS for 6 hours, the lung tissues were collected and homogenized for the extraction of total proteins. The expressions of IκB-α (A, C) and phosphorylation of NF-κB p65 (B, D) were detected by Western blot. The experiment was repeated for 3 times with similar results. Lower panel shows the representative immunoblot and upper panel shows the quantitative analysis of the protein expression. Data are expressed as mean ± SEM; n = 3; * P

    Article Snippet: Antibodies, Reagents and Mice Rabbit anti-NF-κB-p65 (phospho-p65), rabbit anti-IκB-α, mouse monoclonal anti-GAPDH antibodies were bought from Cell Signaling Technology.

    Techniques: Activation Assay, Mouse Assay, Western Blot, Expressing

    Impaired B cell activation in the HOIP-deficient patient. (A) Flow cytometry analysis of CD80 expression from stimulated PBMCs. PBMCs left unstimulated or stimulated for 3 d as indicated were stained for CD19 and with either an isotype control (red histograms) or an antibody directed against CD80 (blue histograms). CD80 expression values (CD80 median, isotype median) are indicated under the histograms (one experiment). (B) Flow cytometry analysis of CD27/CD38 expression on gated CD19 + cells, upon stimulation of PBMCs for 7 d with CD40L+IL-21 or with medium alone. The gated populations in the figure correspond to CD38 hi and CD27 hi plasmablasts (one experiment). (C) Time course of CD40L-stimulated EBV-immortalized B cells from control, HOIP- (patient), and CD40-deficient (CD40 −/− ) patients, followed by immunoblotting for p-IKK, IKKβ, IκBα, and GAPDH (two independent experiments).

    Journal: The Journal of Experimental Medicine

    Article Title: Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia

    doi: 10.1084/jem.20141130

    Figure Lengend Snippet: Impaired B cell activation in the HOIP-deficient patient. (A) Flow cytometry analysis of CD80 expression from stimulated PBMCs. PBMCs left unstimulated or stimulated for 3 d as indicated were stained for CD19 and with either an isotype control (red histograms) or an antibody directed against CD80 (blue histograms). CD80 expression values (CD80 median, isotype median) are indicated under the histograms (one experiment). (B) Flow cytometry analysis of CD27/CD38 expression on gated CD19 + cells, upon stimulation of PBMCs for 7 d with CD40L+IL-21 or with medium alone. The gated populations in the figure correspond to CD38 hi and CD27 hi plasmablasts (one experiment). (C) Time course of CD40L-stimulated EBV-immortalized B cells from control, HOIP- (patient), and CD40-deficient (CD40 −/− ) patients, followed by immunoblotting for p-IKK, IKKβ, IκBα, and GAPDH (two independent experiments).

    Article Snippet: The antibodies used for detection on immunoblots were anti-HOIL-1 N-ter, provided by H. Walczak (University College London, London, England, UK; ), anti-HOIL-1 C-ter provided by K. Iwai (Graduate School of Medicine, Kyoto University, Kyoto, Japan; ), anti-HOIP (PAB6229; Abnova), and anti-linear polyubiquitin chains 1F113F5/Y102L provided by Genentech , anti-NEMO (sc-8330; Santa Cruz Biotechnology, Inc.; #611306; BD), anti-SHARPIN (14626–1-AP; ProteinTech), anti-IκBα (#610690; BD), anti-phospho-IKKα-β (ser176/180; 16A6; Cell Signaling Technology), anti-IKKβ (AM8109a, Abgent), anti-β-tubulin (T4026; Sigma-Aldrich), and anti-GAPDH (sc-365062; Santa Cruz Biotechnology, Inc.) antibodies.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing, Staining

    Impaired TNF- and IL-1β-induced NF-κB activation in the HOIP-deficient patient. (A) Time-course of IL-1β–stimulated (left) or TNF-stimulated (right) SV40-immortalized fibroblasts from control, HOIP-, and HOIL-1–deficient patients, with immunoblotting for phospho-IKK (p-IKK), IKKβ, IκBα, NEMO, and β-tubulin. (B) IL-6 production upon stimulation with TNF and IL-1β in SV-40 fibroblasts from two controls, HOIP- (Patient), HOIL-1–, and NEMO-deficient patients (errors bars indicate the SEM; 3 independent experiments). Unpaired Student’s t tests were performed for the statistical analysis (**, P

    Journal: The Journal of Experimental Medicine

    Article Title: Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia

    doi: 10.1084/jem.20141130

    Figure Lengend Snippet: Impaired TNF- and IL-1β-induced NF-κB activation in the HOIP-deficient patient. (A) Time-course of IL-1β–stimulated (left) or TNF-stimulated (right) SV40-immortalized fibroblasts from control, HOIP-, and HOIL-1–deficient patients, with immunoblotting for phospho-IKK (p-IKK), IKKβ, IκBα, NEMO, and β-tubulin. (B) IL-6 production upon stimulation with TNF and IL-1β in SV-40 fibroblasts from two controls, HOIP- (Patient), HOIL-1–, and NEMO-deficient patients (errors bars indicate the SEM; 3 independent experiments). Unpaired Student’s t tests were performed for the statistical analysis (**, P

    Article Snippet: The antibodies used for detection on immunoblots were anti-HOIL-1 N-ter, provided by H. Walczak (University College London, London, England, UK; ), anti-HOIL-1 C-ter provided by K. Iwai (Graduate School of Medicine, Kyoto University, Kyoto, Japan; ), anti-HOIP (PAB6229; Abnova), and anti-linear polyubiquitin chains 1F113F5/Y102L provided by Genentech , anti-NEMO (sc-8330; Santa Cruz Biotechnology, Inc.; #611306; BD), anti-SHARPIN (14626–1-AP; ProteinTech), anti-IκBα (#610690; BD), anti-phospho-IKKα-β (ser176/180; 16A6; Cell Signaling Technology), anti-IKKβ (AM8109a, Abgent), anti-β-tubulin (T4026; Sigma-Aldrich), and anti-GAPDH (sc-365062; Santa Cruz Biotechnology, Inc.) antibodies.

    Techniques: Activation Assay

    Complementation of impaired NF-κB activation by reintroduction of the WT HOIP allele. (A) Immunoblot of the three LUBAC subunits in SV-40–immortalized fibroblasts from control, HOIP-deficient (Patient), and HOIL-1–deficient cells expressing empty vector (-) or WT-V5 HOIP constructs (+). (B) Time course of IL-1β−stimulated (top) or TNF-stimulated (bottom) SV-40–immortalized fibroblasts from control, HOIP-deficient (Patient-empty), and HOIP-deficient cells complemented with WT HOIP (Patient-HOIP), with immunoblotting for p-IKK, IKKβ, IκBα, and GAPDH. (C) IL-6 production in SV-40 fibroblasts from control, HOIP- (Patient), or HOIL-1–deficient patients without (-) and with (+) WT HOIP complementation, after stimulation with TNF or IL-1β. Errors bars represent the SEM (five independent experiments). Unpaired Student’s t t ests were used for the statistical analysis (**, P

    Journal: The Journal of Experimental Medicine

    Article Title: Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia

    doi: 10.1084/jem.20141130

    Figure Lengend Snippet: Complementation of impaired NF-κB activation by reintroduction of the WT HOIP allele. (A) Immunoblot of the three LUBAC subunits in SV-40–immortalized fibroblasts from control, HOIP-deficient (Patient), and HOIL-1–deficient cells expressing empty vector (-) or WT-V5 HOIP constructs (+). (B) Time course of IL-1β−stimulated (top) or TNF-stimulated (bottom) SV-40–immortalized fibroblasts from control, HOIP-deficient (Patient-empty), and HOIP-deficient cells complemented with WT HOIP (Patient-HOIP), with immunoblotting for p-IKK, IKKβ, IκBα, and GAPDH. (C) IL-6 production in SV-40 fibroblasts from control, HOIP- (Patient), or HOIL-1–deficient patients without (-) and with (+) WT HOIP complementation, after stimulation with TNF or IL-1β. Errors bars represent the SEM (five independent experiments). Unpaired Student’s t t ests were used for the statistical analysis (**, P

    Article Snippet: The antibodies used for detection on immunoblots were anti-HOIL-1 N-ter, provided by H. Walczak (University College London, London, England, UK; ), anti-HOIL-1 C-ter provided by K. Iwai (Graduate School of Medicine, Kyoto University, Kyoto, Japan; ), anti-HOIP (PAB6229; Abnova), and anti-linear polyubiquitin chains 1F113F5/Y102L provided by Genentech , anti-NEMO (sc-8330; Santa Cruz Biotechnology, Inc.; #611306; BD), anti-SHARPIN (14626–1-AP; ProteinTech), anti-IκBα (#610690; BD), anti-phospho-IKKα-β (ser176/180; 16A6; Cell Signaling Technology), anti-IKKβ (AM8109a, Abgent), anti-β-tubulin (T4026; Sigma-Aldrich), and anti-GAPDH (sc-365062; Santa Cruz Biotechnology, Inc.) antibodies.

    Techniques: Activation Assay, Expressing, Plasmid Preparation, Construct