iκbα Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cell Signaling Technology Inc iκbα
    Determination of COS-induced signaling pathways. RAW 264.7 cells were stimulated with 100 μg/mL COS (1874 Da) for the indicated times (0–360 min). Then, cell proteins were extracted and subjected to Western blotting. The effects of COS (1874 Da) on the NF-κB p65 ( A ), MAPK ( B ), IKKα/β and <t>IκBα</t> ( C ), PI3K-Akt ( D ) signaling pathways were determined. The relative expression levels of protein were normalized by β-actin. ( E ) NO production in RAW 264.7 cells pretreated with or without inhibitors after stimulated with 100 μg/mL COS. Data are presented as mean ± SD. (* p
    Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iκbα/product/Cell Signaling Technology Inc
    Average 99 stars, based on 6692 article reviews
    Price from $9.99 to $1999.99
    iκbα - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    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 2178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti iκbα/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2178 article reviews
    Price from $9.99 to $1999.99
    anti iκbα - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc p iκbα
    The anti-inflammatory and anti-fibrotic effects of 5-MTP in vitro. a , b The protein expression and relative quantitative data of <t>IκBα,</t> p-IκBα, NF-κB p65, COX-2 and MCP-1 as well as Nrf2, HO-1 and NQO-1 in HK-2 cells induced by TGF-β1 as indicated. * P
    P Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p iκbα/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2061 article reviews
    Price from $9.99 to $1999.99
    p iκbα - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    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 1750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti iκbα/product/Santa Cruz Biotechnology
    Average 93 stars, based on 1750 article reviews
    Price from $9.99 to $1999.99
    anti iκbα - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    99
    Abcam iκbα
    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of <t>IκBα</t> and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P
    Iκbα, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/iκbα/product/Abcam
    Average 99 stars, based on 690 article reviews
    Price from $9.99 to $1999.99
    iκbα - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/rabbit anti iκbα/product/Cell Signaling Technology Inc
    Average 91 stars, based on 239 article reviews
    Price from $9.99 to $1999.99
    rabbit anti iκbα - by Bioz Stars, 2020-10
    91/100 stars
      Buy from Supplier

    Image Search Results


    Determination of COS-induced signaling pathways. RAW 264.7 cells were stimulated with 100 μg/mL COS (1874 Da) for the indicated times (0–360 min). Then, cell proteins were extracted and subjected to Western blotting. The effects of COS (1874 Da) on the NF-κB p65 ( A ), MAPK ( B ), IKKα/β and IκBα ( C ), PI3K-Akt ( D ) signaling pathways were determined. The relative expression levels of protein were normalized by β-actin. ( E ) NO production in RAW 264.7 cells pretreated with or without inhibitors after stimulated with 100 μg/mL COS. Data are presented as mean ± SD. (* p

    Journal: Marine Drugs

    Article Title: Immunostimulatory Effects of Chitooligosaccharides on RAW 264.7 Mouse Macrophages via Regulation of the MAPK and PI3K/Akt Signaling Pathways

    doi: 10.3390/md17010036

    Figure Lengend Snippet: Determination of COS-induced signaling pathways. RAW 264.7 cells were stimulated with 100 μg/mL COS (1874 Da) for the indicated times (0–360 min). Then, cell proteins were extracted and subjected to Western blotting. The effects of COS (1874 Da) on the NF-κB p65 ( A ), MAPK ( B ), IKKα/β and IκBα ( C ), PI3K-Akt ( D ) signaling pathways were determined. The relative expression levels of protein were normalized by β-actin. ( E ) NO production in RAW 264.7 cells pretreated with or without inhibitors after stimulated with 100 μg/mL COS. Data are presented as mean ± SD. (* p

    Article Snippet: Primary antibodies to TNF-α, cyclooxygenase (COX)-2, p85, PDK1, Akt, IκBα, p65, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, and phospho-specific antibodies to p85, PDK1, Akt, IκBα, p65, ERK, JNK, p38 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA) except the primary antibody against inducible NO synthase (iNOS) from Abcam (Cambridge, MA, USA).

    Techniques: Western Blot, Expressing

    Puerarin-V attenuated LPS-induced inflammation in HCAECs associated with the PPAR-γ/NF-κB pathway. ( A , B ) The mRNA levels of TNF-α and IL-1β. ( C ) Confluent cultures of HCAEC were preincubated for 2 h with puerarin-V (3 μM). Thereafter, different concentrations of LPS were added and cells were cultivated for 24 h. The black bars show the cultures without puerarin-V treatment; the blue bars show the puerarin-V treated cultures. ( D ) Confluent cultures of HCAEC were preincubated for 2 h with different amounts of puerarin-V (μM). Thereafter, LPS (100 ng/ml) was added and cells were cultivated for 24 h. ( E ) PPAR-γ expression. ( F ) p-NF-κB/NF-κB expression. ( G ) p-IκB-α/IκB-α expression. ( H ) p-IKKα/β/IKKα/β expression. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM ( n = 4–6). ## p

    Journal: Molecules

    Article Title: Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway

    doi: 10.3390/molecules23123322

    Figure Lengend Snippet: Puerarin-V attenuated LPS-induced inflammation in HCAECs associated with the PPAR-γ/NF-κB pathway. ( A , B ) The mRNA levels of TNF-α and IL-1β. ( C ) Confluent cultures of HCAEC were preincubated for 2 h with puerarin-V (3 μM). Thereafter, different concentrations of LPS were added and cells were cultivated for 24 h. The black bars show the cultures without puerarin-V treatment; the blue bars show the puerarin-V treated cultures. ( D ) Confluent cultures of HCAEC were preincubated for 2 h with different amounts of puerarin-V (μM). Thereafter, LPS (100 ng/ml) was added and cells were cultivated for 24 h. ( E ) PPAR-γ expression. ( F ) p-NF-κB/NF-κB expression. ( G ) p-IκB-α/IκB-α expression. ( H ) p-IKKα/β/IKKα/β expression. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM ( n = 4–6). ## p

    Article Snippet: The antibodies against P-NF-κBp65, NF-κBp65, p-IκB-α, IκB-α, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    Puerarin-V attenuated ISO-induced inflammation in the MI mice associated with the PPAR-γ/NF-κB pathway: ( A ) PPAR-γ; ( B ) p-NF-κB/NF-κB; ( C ) p-IκB-α/IκB-α; and ( D ) p-IKKα/β/IKKα/β. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM ( n = 6). ### p

    Journal: Molecules

    Article Title: Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway

    doi: 10.3390/molecules23123322

    Figure Lengend Snippet: Puerarin-V attenuated ISO-induced inflammation in the MI mice associated with the PPAR-γ/NF-κB pathway: ( A ) PPAR-γ; ( B ) p-NF-κB/NF-κB; ( C ) p-IκB-α/IκB-α; and ( D ) p-IKKα/β/IKKα/β. Relative density analysis of the protein bands was shown by the western blot with GAPDH as control. Data are shown as mean ± SEM ( n = 6). ### p

    Article Snippet: The antibodies against P-NF-κBp65, NF-κBp65, p-IκB-α, IκB-α, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Mouse Assay, Western Blot

    Gal-8 activity in OA chondrocytes is mediated by the NF-κB signaling pathway. a – c Quantitative WB analyses were performed with proteins isolated from OA chondrocytes after overnight starvation and treatment with 10 µg/ml Gal-8S for 15 min, 1, 4, and 16 h. a Shown are blots of one representative patient for phosphorylated IκBα, IκBα, phosphorylated p65, p65, and α-tubulin. b – c Over-time ratios are presented between phosphorylated IκBα and IκBα ( b ), as well as phosphorylated p65 and p65 ( c ). Data were normalized for α-tubulin and expressed as relative quantity compared to the untreated control set to 1 ( n = 2 patients; * p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Galectin-8 induces functional disease markers in human osteoarthritis and cooperates with galectins-1 and -3

    doi: 10.1007/s00018-018-2856-2

    Figure Lengend Snippet: Gal-8 activity in OA chondrocytes is mediated by the NF-κB signaling pathway. a – c Quantitative WB analyses were performed with proteins isolated from OA chondrocytes after overnight starvation and treatment with 10 µg/ml Gal-8S for 15 min, 1, 4, and 16 h. a Shown are blots of one representative patient for phosphorylated IκBα, IκBα, phosphorylated p65, p65, and α-tubulin. b – c Over-time ratios are presented between phosphorylated IκBα and IκBα ( b ), as well as phosphorylated p65 and p65 ( c ). Data were normalized for α-tubulin and expressed as relative quantity compared to the untreated control set to 1 ( n = 2 patients; * p

    Article Snippet: After blocking with 5% milk/PBS, membranes were incubated for 2 h with primary antibodies: anti-Gal-8 (rabbit polyclonal), anti-phospho IκBα (Ser32/36, 1:1000, mouse monoclonal; Cell Signaling), anti-IκBα (1:1000, rabbit monoclonal; Cell Signaling), anti-phospho NF-κB p65 (Ser536; 1:1000; rabbit monoclonal; Cell Signaling), anti-NF-κB p65 (1:1000; mouse monoclonal; Cell Signaling) and anti-α-tubulin (1:1000; mouse monoclonal; Cell Signaling).

    Techniques: Activity Assay, Western Blot, Isolation

    Effects of As 2 O 3 , GSPE on the protein expression of p-IκB-α, IκB-α, IKKα/β, NF-κBp65, and NF-κBp50 in BEAS-2B cells. The BEAS-2B cells were treated with 5 μmol/L As 2 O 3 (n = 3). GSPE (50 mg/L) was used to antagonize the effect of arsenic. The expression of the various proteins was detected by Western blot. The levels of p-IκB-α (a), IκB-α (b), IKKα/β (c), NF-κBp65 (d), and NF-κBp50 (e) (without BAY 11–7082), respectively. The Image J software was used to measure the gray value of each blot and β-actin was used as a loading control. Values are expressed as means ± SD. a P

    Journal: Experimental Biology and Medicine

    Article Title: Grape seed proanthocyanidin extract alleviates arsenic-induced lung damage through NF-κB signaling

    doi: 10.1177/1535370219829881

    Figure Lengend Snippet: Effects of As 2 O 3 , GSPE on the protein expression of p-IκB-α, IκB-α, IKKα/β, NF-κBp65, and NF-κBp50 in BEAS-2B cells. The BEAS-2B cells were treated with 5 μmol/L As 2 O 3 (n = 3). GSPE (50 mg/L) was used to antagonize the effect of arsenic. The expression of the various proteins was detected by Western blot. The levels of p-IκB-α (a), IκB-α (b), IKKα/β (c), NF-κBp65 (d), and NF-κBp50 (e) (without BAY 11–7082), respectively. The Image J software was used to measure the gray value of each blot and β-actin was used as a loading control. Values are expressed as means ± SD. a P

    Article Snippet: Western blot was performed as previously described for the detection of p-IκB-α (9246; Cell Signaling, Danvers, MA, USA), IκB-α (4814; Cell Signaling, Danvers, MA, USA), IKKα/β (ab178870; Abcam, Cambridge, MA, USA), NF-κBp65 (D14E12; Cell Signaling, Danvers, MA, USA), and NF-κBp50 (D4P4D; Cell Signaling, Danvers, MA, USA). β-actin was used as a loading control.

    Techniques: Expressing, Western Blot, Software

    Effects of arsenic and GSPE on mRNA expression of IκB-α, IKKα, IKKβ, NF-κBp65, and NF-κBp50 in lung tissue induced by arsenic in mice. Mice were exposed to As 2 O 3 (4 mg/kg) (n = 10); 400 mg/kg GSPE was used to antagonize the effects of arsenic. The expression of each mRNA in the control, GSPE, AsO 3 , and As 2 O 3 +GSPE groups was detected by RT-PCR. mRNA levels of IκB-α (a), IKKα (b), IKKβ (c), NF-κBp65 (d), and NF-κBp50 (E), respectively. Values are expressed as means ± SD. a P

    Journal: Experimental Biology and Medicine

    Article Title: Grape seed proanthocyanidin extract alleviates arsenic-induced lung damage through NF-κB signaling

    doi: 10.1177/1535370219829881

    Figure Lengend Snippet: Effects of arsenic and GSPE on mRNA expression of IκB-α, IKKα, IKKβ, NF-κBp65, and NF-κBp50 in lung tissue induced by arsenic in mice. Mice were exposed to As 2 O 3 (4 mg/kg) (n = 10); 400 mg/kg GSPE was used to antagonize the effects of arsenic. The expression of each mRNA in the control, GSPE, AsO 3 , and As 2 O 3 +GSPE groups was detected by RT-PCR. mRNA levels of IκB-α (a), IKKα (b), IKKβ (c), NF-κBp65 (d), and NF-κBp50 (E), respectively. Values are expressed as means ± SD. a P

    Article Snippet: Western blot was performed as previously described for the detection of p-IκB-α (9246; Cell Signaling, Danvers, MA, USA), IκB-α (4814; Cell Signaling, Danvers, MA, USA), IKKα/β (ab178870; Abcam, Cambridge, MA, USA), NF-κBp65 (D14E12; Cell Signaling, Danvers, MA, USA), and NF-κBp50 (D4P4D; Cell Signaling, Danvers, MA, USA). β-actin was used as a loading control.

    Techniques: Expressing, Mouse Assay, Allele-specific Oligonucleotide, Reverse Transcription Polymerase Chain Reaction

    Effects of GSPE on the protein expression of p-IκB-α, IκB-α, IKKα/β, NF-κBp65, and NF-κBp50 in lung tissues induced by arsenic. Mice were exposed to As 2 O 3 (4 mg/kg) (n = 10); 400 mg/kg GSPE was used to antagonize the effects of arsenic. The expression of each protein in the control, GSPE, AsO 3 , and As 2 O 3 +GSPE groups was detected by Western blot. Protein levels of p-IκB-α (a), IκB-α (b), IKKα/β (c), NF-κBp65 (d), and NF-κBp50 (E), respectively. The Image J software was used to measure the gray value of each blot and β-actin was used as a loading control. Values are expressed as means ± SD. a P

    Journal: Experimental Biology and Medicine

    Article Title: Grape seed proanthocyanidin extract alleviates arsenic-induced lung damage through NF-κB signaling

    doi: 10.1177/1535370219829881

    Figure Lengend Snippet: Effects of GSPE on the protein expression of p-IκB-α, IκB-α, IKKα/β, NF-κBp65, and NF-κBp50 in lung tissues induced by arsenic. Mice were exposed to As 2 O 3 (4 mg/kg) (n = 10); 400 mg/kg GSPE was used to antagonize the effects of arsenic. The expression of each protein in the control, GSPE, AsO 3 , and As 2 O 3 +GSPE groups was detected by Western blot. Protein levels of p-IκB-α (a), IκB-α (b), IKKα/β (c), NF-κBp65 (d), and NF-κBp50 (E), respectively. The Image J software was used to measure the gray value of each blot and β-actin was used as a loading control. Values are expressed as means ± SD. a P

    Article Snippet: Western blot was performed as previously described for the detection of p-IκB-α (9246; Cell Signaling, Danvers, MA, USA), IκB-α (4814; Cell Signaling, Danvers, MA, USA), IKKα/β (ab178870; Abcam, Cambridge, MA, USA), NF-κBp65 (D14E12; Cell Signaling, Danvers, MA, USA), and NF-κBp50 (D4P4D; Cell Signaling, Danvers, MA, USA). β-actin was used as a loading control.

    Techniques: Expressing, Mouse Assay, Allele-specific Oligonucleotide, Western Blot, Software

    Effects of As 2 O 3 , GSPE, and BAY 11–7082 on the protein expression of p-IκB-α, IκB-α, IKKα/β, NF-κBp65, and NF-κBp50 in BEAS-2B cells. The BEAS-2B cells were treated with 5 μmol/L As 2 O 3 (n = 3). GSPE (50 mg/L) was used to antagonize the effect of arsenic. The expression of the various proteins was detected by Western blot. The levels of p-IκB-α (a), IκB-α (b), IKKα/β (c), NF-κBp65 (d), and NF-κBp50 (e) (with BAY 11–7082), respectively. The Image J software was used to measure the gray value of each blot and β-actin was used as a loading control. Values are expressed as means ± SD. a P

    Journal: Experimental Biology and Medicine

    Article Title: Grape seed proanthocyanidin extract alleviates arsenic-induced lung damage through NF-κB signaling

    doi: 10.1177/1535370219829881

    Figure Lengend Snippet: Effects of As 2 O 3 , GSPE, and BAY 11–7082 on the protein expression of p-IκB-α, IκB-α, IKKα/β, NF-κBp65, and NF-κBp50 in BEAS-2B cells. The BEAS-2B cells were treated with 5 μmol/L As 2 O 3 (n = 3). GSPE (50 mg/L) was used to antagonize the effect of arsenic. The expression of the various proteins was detected by Western blot. The levels of p-IκB-α (a), IκB-α (b), IKKα/β (c), NF-κBp65 (d), and NF-κBp50 (e) (with BAY 11–7082), respectively. The Image J software was used to measure the gray value of each blot and β-actin was used as a loading control. Values are expressed as means ± SD. a P

    Article Snippet: Western blot was performed as previously described for the detection of p-IκB-α (9246; Cell Signaling, Danvers, MA, USA), IκB-α (4814; Cell Signaling, Danvers, MA, USA), IKKα/β (ab178870; Abcam, Cambridge, MA, USA), NF-κBp65 (D14E12; Cell Signaling, Danvers, MA, USA), and NF-κBp50 (D4P4D; Cell Signaling, Danvers, MA, USA). β-actin was used as a loading control.

    Techniques: Expressing, Western Blot, Software

    Effects of As 2 O 3 , GSPE, and BAY 11–7082 on the mRNA expression of IκB-α, IKKα, IKKβ, NF-κBp65, and NF-κBp50 in BEAS-2B cells. The BEAS-2B cells were treated with 5 μmol/L As 2 O 3 (n = 10) and GSPE (50 mg/L) and BAY 11–7082 (50 mg/L) were used to antagonize the effects of arsenic. The relative mRNA levels were determined by RT-PCR using gene-specific primers. Values are expressed as means ± SD. a P

    Journal: Experimental Biology and Medicine

    Article Title: Grape seed proanthocyanidin extract alleviates arsenic-induced lung damage through NF-κB signaling

    doi: 10.1177/1535370219829881

    Figure Lengend Snippet: Effects of As 2 O 3 , GSPE, and BAY 11–7082 on the mRNA expression of IκB-α, IKKα, IKKβ, NF-κBp65, and NF-κBp50 in BEAS-2B cells. The BEAS-2B cells were treated with 5 μmol/L As 2 O 3 (n = 10) and GSPE (50 mg/L) and BAY 11–7082 (50 mg/L) were used to antagonize the effects of arsenic. The relative mRNA levels were determined by RT-PCR using gene-specific primers. Values are expressed as means ± SD. a P

    Article Snippet: Western blot was performed as previously described for the detection of p-IκB-α (9246; Cell Signaling, Danvers, MA, USA), IκB-α (4814; Cell Signaling, Danvers, MA, USA), IKKα/β (ab178870; Abcam, Cambridge, MA, USA), NF-κBp65 (D14E12; Cell Signaling, Danvers, MA, USA), and NF-κBp50 (D4P4D; Cell Signaling, Danvers, MA, USA). β-actin was used as a loading control.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Procyanidin suppressed the NF-κB signaling pathway in THP-1 cells. THP-1 cells were treated with different concentrations of procyanidin (0.3, 1, 3, 10 µM) in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml) for 30 min. (A) Protein levels of p-p65, p65, p-IKKα/β, IKKα, IKKβ, p-IκBα, and IκBα were determined by western blot. (B) Subcellular localization of p-p65 was examined by immunofluorescence by a confocal microscope. (C–E) mRNA levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were measured 6 h after LPS stimulation by real-time PCR. ## P

    Journal: Frontiers in Immunology

    Article Title: The Antioxidant Procyanidin Reduces Reactive Oxygen Species Signaling in Macrophages and Ameliorates Experimental Colitis in Mice

    doi: 10.3389/fimmu.2017.01910

    Figure Lengend Snippet: Procyanidin suppressed the NF-κB signaling pathway in THP-1 cells. THP-1 cells were treated with different concentrations of procyanidin (0.3, 1, 3, 10 µM) in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml) for 30 min. (A) Protein levels of p-p65, p65, p-IKKα/β, IKKα, IKKβ, p-IκBα, and IκBα were determined by western blot. (B) Subcellular localization of p-p65 was examined by immunofluorescence by a confocal microscope. (C–E) mRNA levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were measured 6 h after LPS stimulation by real-time PCR. ## P

    Article Snippet: Antibodies for NLRP3, phospho-p65, total p65, phospho-IKKα/β, total IKKα, anti-total IKKβ, phospho-IκBα, total IκBα and caspase-1 were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot, Immunofluorescence, Microscopy, Real-time Polymerase Chain Reaction

    Hypertonicity stimulation of IκB-α phosphorylation in tonicity and in a time-dependent manner. ( A ) Cells were exposed to 300, 375, and 450 mOsm media for 1 hour. Specificity of IκB-α phosphorylation was validated with pretreatment

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TRPV1 Activation Is Required for Hypertonicity-Stimulated Inflammatory Cytokine Release in Human Corneal Epithelial Cells

    doi: 10.1167/iovs.10-5801

    Figure Lengend Snippet: Hypertonicity stimulation of IκB-α phosphorylation in tonicity and in a time-dependent manner. ( A ) Cells were exposed to 300, 375, and 450 mOsm media for 1 hour. Specificity of IκB-α phosphorylation was validated with pretreatment

    Article Snippet: Anti–phospho-p38 and phospho-IκB-α were from Cell Signaling Technology (Danvers, MA).

    Techniques:

    Effects of modulation of TRPV1, EGFR, ERK, and p38 on hypertonicity-induced IκB-α phosphorylation. Cells were pretreated for 30 minutes with capsazepine (10 μM), AG 1478 (10 μM), PD 98059 (10 μM), or SB 203580 (10

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: TRPV1 Activation Is Required for Hypertonicity-Stimulated Inflammatory Cytokine Release in Human Corneal Epithelial Cells

    doi: 10.1167/iovs.10-5801

    Figure Lengend Snippet: Effects of modulation of TRPV1, EGFR, ERK, and p38 on hypertonicity-induced IκB-α phosphorylation. Cells were pretreated for 30 minutes with capsazepine (10 μM), AG 1478 (10 μM), PD 98059 (10 μM), or SB 203580 (10

    Article Snippet: Anti–phospho-p38 and phospho-IκB-α were from Cell Signaling Technology (Danvers, MA).

    Techniques:

    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

    The anti-inflammatory and anti-fibrotic effects of 5-MTP in vitro. a , b The protein expression and relative quantitative data of IκBα, p-IκBα, NF-κB p65, COX-2 and MCP-1 as well as Nrf2, HO-1 and NQO-1 in HK-2 cells induced by TGF-β1 as indicated. * P

    Journal: Nature Communications

    Article Title: Identification of serum metabolites associating with chronic kidney disease progression and anti-fibrotic effect of 5-methoxytryptophan

    doi: 10.1038/s41467-019-09329-0

    Figure Lengend Snippet: The anti-inflammatory and anti-fibrotic effects of 5-MTP in vitro. a , b The protein expression and relative quantitative data of IκBα, p-IκBα, NF-κB p65, COX-2 and MCP-1 as well as Nrf2, HO-1 and NQO-1 in HK-2 cells induced by TGF-β1 as indicated. * P

    Article Snippet: The following primary antibodies were employed (dilution): collagen I (1:5000, ab34710, Abcam, USA), α-SMA (1:300, ab7817, Abcam, USA), fibronectin (1:1000, ab2413, Abcam, USA), vimentin (1:1000, ab92547, Abcam, USA), E-cadherin (1:500, ab76055, Abcam, USA), p-IκBα (1:2000, 2859, Cell Signaling Technology, USA), IκBα (1:2000, 4812, Cell Signaling Technology, USA), NF-κB p65 (1:1000, ab16502, Abcam, USA), MCP-1 (1:1000, ab7202, Abcam, USA), COX-2 (1:1000, ab62331, Abcam, USA), Nrf2 (1:1000, ab31163, Abcam, USA), HO-1 (1:1000, ab68477, Abcam, USA), NQO-1 (1:1000, ab28947, Abcam, USA) and TPH-1 (1:500, ab52954, Abcam, USA).

    Techniques: In Vitro, Expressing

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    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 membranes were used in Western blot analysis with polyclonal rabbit antibody directed against IκBα (Santa Cruz Biotechnology, Inc.; SC-371) at a dilution of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.; SC-230) at a dilution of 1:5,000 before incubation with a second antibody and development with enhanced chemiluminescence.

    Techniques: Construct

    Molecular effects of selinexor treatment on HGG cells: a. NGFR expression by qPCR following vehicle or selinexor treatment (16h) at indicated dose levels (n=3); b. total NGFR protein expression following vehicle or selinexor treatment; c. cell surface NGFR protein expression; d. plot of NGFR cell surface expression versus selinexor IC 50 in DMG cell lines; e. nuclear IκB-α protein expression following vehicle or selinexor treatment; f. nuclear NF-κB (p65) protein expression following vehicle or selinexor treatment; g. ratio of nuclear NF-κB/IκB-α from panels e and f; h. nuclear p-NF-κB (S536) protein expression following vehicle or selinexor treatment; i. ratio of nuclear p-NF-κB (S536)/NF-κB from panels f and h. Panels c – f are quantification of immunofluorescence images following 16h selinexor treatment at 5xIC 50 dose by cell line (n = number of cells quantified *p

    Journal: Molecular cancer therapeutics

    Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

    doi: 10.1158/1535-7163.MCT-18-1319

    Figure Lengend Snippet: Molecular effects of selinexor treatment on HGG cells: a. NGFR expression by qPCR following vehicle or selinexor treatment (16h) at indicated dose levels (n=3); b. total NGFR protein expression following vehicle or selinexor treatment; c. cell surface NGFR protein expression; d. plot of NGFR cell surface expression versus selinexor IC 50 in DMG cell lines; e. nuclear IκB-α protein expression following vehicle or selinexor treatment; f. nuclear NF-κB (p65) protein expression following vehicle or selinexor treatment; g. ratio of nuclear NF-κB/IκB-α from panels e and f; h. nuclear p-NF-κB (S536) protein expression following vehicle or selinexor treatment; i. ratio of nuclear p-NF-κB (S536)/NF-κB from panels f and h. Panels c – f are quantification of immunofluorescence images following 16h selinexor treatment at 5xIC 50 dose by cell line (n = number of cells quantified *p

    Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence

    In vitro effects of selinexor treatment: total NGFR, nuclear IκB-α and NF-κB (p65) from immunofluorescence images of mouse PDX tumor samples (n = number of cells quantified *p

    Journal: Molecular cancer therapeutics

    Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

    doi: 10.1158/1535-7163.MCT-18-1319

    Figure Lengend Snippet: In vitro effects of selinexor treatment: total NGFR, nuclear IκB-α and NF-κB (p65) from immunofluorescence images of mouse PDX tumor samples (n = number of cells quantified *p

    Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

    Techniques: In Vitro, Immunofluorescence

    Molecular and proliferative effects of NGFR KD: a. NGFR gene expression levels in DIPG6 and SF7761 shNull (empty vector control) and shNGFR ( NGFR KD) cells (n=3); b. ratio of RELA/NFKBIA gene expression in SF7761 shNull and shNGFR cells (n=3); c. nuclear IκB-α and NF-κB (p65) protein levels by immunofluorescence and NF-κB (p65)/IκB-α in SF7761 shNull and shNGFR cells (n=number of cells quantified); d. week over week expansion rate for DIPG6 and SF7761 shNull and shNGFR (n=4 for all samples).

    Journal: Molecular cancer therapeutics

    Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

    doi: 10.1158/1535-7163.MCT-18-1319

    Figure Lengend Snippet: Molecular and proliferative effects of NGFR KD: a. NGFR gene expression levels in DIPG6 and SF7761 shNull (empty vector control) and shNGFR ( NGFR KD) cells (n=3); b. ratio of RELA/NFKBIA gene expression in SF7761 shNull and shNGFR cells (n=3); c. nuclear IκB-α and NF-κB (p65) protein levels by immunofluorescence and NF-κB (p65)/IκB-α in SF7761 shNull and shNGFR cells (n=number of cells quantified); d. week over week expansion rate for DIPG6 and SF7761 shNull and shNGFR (n=4 for all samples).

    Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

    Techniques: Expressing, Plasmid Preparation, Immunofluorescence

    Effects of selinexor treatment on NGFR KD and NGFR overexpressing cells: a. NGFR gene expression in SF7761 shNull and shNGFR KD cells (n=3) following selinexor treatment for 16h at indicated dose levels; b. selinexor IC 50 levels in SF7761 shNull and shNGFR cells (n=3); c. apoptosis levels by caspase 3/7 luminescence in SF7761 shNull and shNGFR cells at 24h and 48h time points (n=3); d. log 10 of relative transcriptionally active NF-κB by ELISA in SF7761 shNull and shNGFR cells following selinexor treatment for 16h at 1μM dose level (n=3); e. relative gene expression levels of NGFR, NFKBIA and RELA by qPCR (n=3) and protein expression levels of total NGFR and nuclear NF-κB and IκB-α in cells transfected with empty vector or NGFR cDNA; f. selinexor IC 50 levels in DIPG4 wild type or cells transfected with IκB-α super repressor (n=3); g. selinexor IC 50 levels in DIPG4 shNull, shNGFR and shNGFR cells transfected with IκB-α super repressor (n=3).

    Journal: Molecular cancer therapeutics

    Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

    doi: 10.1158/1535-7163.MCT-18-1319

    Figure Lengend Snippet: Effects of selinexor treatment on NGFR KD and NGFR overexpressing cells: a. NGFR gene expression in SF7761 shNull and shNGFR KD cells (n=3) following selinexor treatment for 16h at indicated dose levels; b. selinexor IC 50 levels in SF7761 shNull and shNGFR cells (n=3); c. apoptosis levels by caspase 3/7 luminescence in SF7761 shNull and shNGFR cells at 24h and 48h time points (n=3); d. log 10 of relative transcriptionally active NF-κB by ELISA in SF7761 shNull and shNGFR cells following selinexor treatment for 16h at 1μM dose level (n=3); e. relative gene expression levels of NGFR, NFKBIA and RELA by qPCR (n=3) and protein expression levels of total NGFR and nuclear NF-κB and IκB-α in cells transfected with empty vector or NGFR cDNA; f. selinexor IC 50 levels in DIPG4 wild type or cells transfected with IκB-α super repressor (n=3); g. selinexor IC 50 levels in DIPG4 shNull, shNGFR and shNGFR cells transfected with IκB-α super repressor (n=3).

    Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

    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

    Selective activation of NOD1 stimulates NF-κB and TGF-β pathways in cardiac fibroblasts. (A) Confocal microscopy images of cardiac fibroblasts isolated from mouse hearts stained for NOD1. (B) Isolated murine cardiac fibroblasts were incubated for 15 to 60 min, 24 h or 48 h with 40 µg/ml iE. iE-treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min), NOS2 (24 h) and COX2 (48 h) protein levels. (C) Treatment of fibroblasts with iE for 72 h promoted an increase of TRβ2, P-Smad/Smad, PAI-1 and FGF-2 protein levels. GAPDH was used to normalize all target protein levels. Data are illustrated in histograms as mean±SEM vs. vehicle (100%; n = 3–5 samples).*p

    Journal: PLoS ONE

    Article Title: NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

    doi: 10.1371/journal.pone.0045260

    Figure Lengend Snippet: Selective activation of NOD1 stimulates NF-κB and TGF-β pathways in cardiac fibroblasts. (A) Confocal microscopy images of cardiac fibroblasts isolated from mouse hearts stained for NOD1. (B) Isolated murine cardiac fibroblasts were incubated for 15 to 60 min, 24 h or 48 h with 40 µg/ml iE. iE-treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min), NOS2 (24 h) and COX2 (48 h) protein levels. (C) Treatment of fibroblasts with iE for 72 h promoted an increase of TRβ2, P-Smad/Smad, PAI-1 and FGF-2 protein levels. GAPDH was used to normalize all target protein levels. Data are illustrated in histograms as mean±SEM vs. vehicle (100%; n = 3–5 samples).*p

    Article Snippet: Antibodies for NOD1, P-RIP2, RIP2, P-IKK, IKK, P-IκBα, IκBα, NOS2, COX2, TRβ1, TRβ2, P-Smad (Smad 2/3), Smad, PAI-1, Bcl-xL, Bax, Bad, X-IAP and FGF-2 were from Santa Cruz Biotech or Cell Signaling Technology.

    Techniques: Activation Assay, Confocal Microscopy, Isolation, Staining, Incubation

    The NOD1 agonist iE induces NF-κB activation and apoptosis in cardiomyocytes and H9c2 cells. Effect of NOD1 ablation with siRNAs. (A) Native murine cardiomyocytes were incubated for 15 to 60 min, or 48 h with 20 µg/ml of iE. iE treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min) and COX2 (48 h) protein levels. (B) iE induces a decrease in cardiomyocyte viability in a dose-response form. Cardiomyocytes incubated for 2 h with 10–30 µg/ml of iE; staurosporine (Stau, 100 ng/ml) were used as a positive control to induce apoptosis. (C). iE treatment induces an up-regulation of caspase 3 and Bax protein levels and down-regulates the BcL-xL and X-IAP protein levels. (D) iE or Stau administration for 24 h in H9c2 promoted a decrease in viability as determined by MTT activity. NOD1 suppression by siRNA prevented the effect of iE on cell viability. (E) Representative blot of NOD1, caspase 3 and GAPDH obtained in vehicle and iE treated cells, NOD1 siRNAs (siNOD1) and NOD1 siRNA+iE in H9c2 cells. All targets protein levels were normalized by GAPDH. Data are expressed in histograms representing the mean±SEM vs . vehicle (100%); n = 3–5 samples per condition.*p

    Journal: PLoS ONE

    Article Title: NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

    doi: 10.1371/journal.pone.0045260

    Figure Lengend Snippet: The NOD1 agonist iE induces NF-κB activation and apoptosis in cardiomyocytes and H9c2 cells. Effect of NOD1 ablation with siRNAs. (A) Native murine cardiomyocytes were incubated for 15 to 60 min, or 48 h with 20 µg/ml of iE. iE treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min) and COX2 (48 h) protein levels. (B) iE induces a decrease in cardiomyocyte viability in a dose-response form. Cardiomyocytes incubated for 2 h with 10–30 µg/ml of iE; staurosporine (Stau, 100 ng/ml) were used as a positive control to induce apoptosis. (C). iE treatment induces an up-regulation of caspase 3 and Bax protein levels and down-regulates the BcL-xL and X-IAP protein levels. (D) iE or Stau administration for 24 h in H9c2 promoted a decrease in viability as determined by MTT activity. NOD1 suppression by siRNA prevented the effect of iE on cell viability. (E) Representative blot of NOD1, caspase 3 and GAPDH obtained in vehicle and iE treated cells, NOD1 siRNAs (siNOD1) and NOD1 siRNA+iE in H9c2 cells. All targets protein levels were normalized by GAPDH. Data are expressed in histograms representing the mean±SEM vs . vehicle (100%); n = 3–5 samples per condition.*p

    Article Snippet: Antibodies for NOD1, P-RIP2, RIP2, P-IKK, IKK, P-IκBα, IκBα, NOS2, COX2, TRβ1, TRβ2, P-Smad (Smad 2/3), Smad, PAI-1, Bcl-xL, Bax, Bad, X-IAP and FGF-2 were from Santa Cruz Biotech or Cell Signaling Technology.

    Techniques: Activation Assay, Incubation, Positive Control, MTT Assay, Activity Assay

    Murine cardiac tissue expresses NOD1. Specific stimulation of NOD1 induces NF-κB pathway activation. (A) Histograms show NOD1 mRNA levels in different mouse tissues. (B) NOD1 protein levels in mouse hearts were analyzed by western-blot. Animals received i.p. 150 µg of iEDAP (iE)/day, a selective agonist of NOD1, or vehicle (Veh.). After 2 weeks of treatment an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα protein ratio (B), higher p65 binding to κB motifs determined by ELISA (C), and NOS2 and COX2 (D) protein levels were observed in iE treated hearts. NOS2 and COX2 protein values were normalized with GAPDH. Representative blots are shown in the left panels and right panels illustrate the histograms representing the mean (band ratio)±SEM values vs. Veh. (100%); n = 4–6 animals. *p

    Journal: PLoS ONE

    Article Title: NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

    doi: 10.1371/journal.pone.0045260

    Figure Lengend Snippet: Murine cardiac tissue expresses NOD1. Specific stimulation of NOD1 induces NF-κB pathway activation. (A) Histograms show NOD1 mRNA levels in different mouse tissues. (B) NOD1 protein levels in mouse hearts were analyzed by western-blot. Animals received i.p. 150 µg of iEDAP (iE)/day, a selective agonist of NOD1, or vehicle (Veh.). After 2 weeks of treatment an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα protein ratio (B), higher p65 binding to κB motifs determined by ELISA (C), and NOS2 and COX2 (D) protein levels were observed in iE treated hearts. NOS2 and COX2 protein values were normalized with GAPDH. Representative blots are shown in the left panels and right panels illustrate the histograms representing the mean (band ratio)±SEM values vs. Veh. (100%); n = 4–6 animals. *p

    Article Snippet: Antibodies for NOD1, P-RIP2, RIP2, P-IKK, IKK, P-IκBα, IκBα, NOS2, COX2, TRβ1, TRβ2, P-Smad (Smad 2/3), Smad, PAI-1, Bcl-xL, Bax, Bad, X-IAP and FGF-2 were from Santa Cruz Biotech or Cell Signaling Technology.

    Techniques: Activation Assay, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

    Effect of NOD1 siRNAs on TGF-β pathway. Proposed role for NOD1 activation in cardiac cells. (A) Representative blot of NOD1, TRβ2, P-Smad/Smad, PAI-1, and GAPDH levels in vehicle, iE, NOD1 siRNAs (siNOD1) and NOD1 siRNA + iE (siNOD1+iE) NIH-3T3 cells. Scrambled siRNA sequences did not modify the basal levels after iE-treatment. (B)The selective agonist iEDAP induces NOD1 activation. Both in cardiomyocytes and cardiac fibroblasts, NOD1 promotes the activation of the NF-κB pathway through IKK/IκBα signaling. NF-κB activation induces a proinflammatory response, including the expression of NOS2 and COX2. Moreover, TGF-β is activated in cardiac fibroblasts resulting in PAI-1 transcriptional activation by TRβ and Smad factors. Finally, the enhancement and activation of Bax and caspases and the downregulation of X-IAP promoted by NOD1 agonist in cardiomyocytes, contributes to elicit an apoptotic response associated with NOD1 activation. Altogether, cardiomyocyte apoptosis and the pro-fibrotic profile observed in cardiac fibroblasts might contribute to the onset of cardiomyopathy linked to proinflammatory disease.

    Journal: PLoS ONE

    Article Title: NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

    doi: 10.1371/journal.pone.0045260

    Figure Lengend Snippet: Effect of NOD1 siRNAs on TGF-β pathway. Proposed role for NOD1 activation in cardiac cells. (A) Representative blot of NOD1, TRβ2, P-Smad/Smad, PAI-1, and GAPDH levels in vehicle, iE, NOD1 siRNAs (siNOD1) and NOD1 siRNA + iE (siNOD1+iE) NIH-3T3 cells. Scrambled siRNA sequences did not modify the basal levels after iE-treatment. (B)The selective agonist iEDAP induces NOD1 activation. Both in cardiomyocytes and cardiac fibroblasts, NOD1 promotes the activation of the NF-κB pathway through IKK/IκBα signaling. NF-κB activation induces a proinflammatory response, including the expression of NOS2 and COX2. Moreover, TGF-β is activated in cardiac fibroblasts resulting in PAI-1 transcriptional activation by TRβ and Smad factors. Finally, the enhancement and activation of Bax and caspases and the downregulation of X-IAP promoted by NOD1 agonist in cardiomyocytes, contributes to elicit an apoptotic response associated with NOD1 activation. Altogether, cardiomyocyte apoptosis and the pro-fibrotic profile observed in cardiac fibroblasts might contribute to the onset of cardiomyopathy linked to proinflammatory disease.

    Article Snippet: Antibodies for NOD1, P-RIP2, RIP2, P-IKK, IKK, P-IκBα, IκBα, NOS2, COX2, TRβ1, TRβ2, P-Smad (Smad 2/3), Smad, PAI-1, Bcl-xL, Bax, Bad, X-IAP and FGF-2 were from Santa Cruz Biotech or Cell Signaling Technology.

    Techniques: Activation Assay, Expressing

    The overexpression of TRADD restores ORF3-inhibited NF-kB signaling. ( A ) TRADD plasmids were co-transfected with ORF3 at a ratio of 1:2 for 48 h, and the cells were then treated with Poly(I:C) for 12 h. Phosphorylated IκBα was assessed to measure NF-κB activity using western blotting, with β-actin serving as a loading control. Overexpression of TRADD restores ORF3-inhibited NF-κB signaling; the relative densitometric ratios for experimental bands are shown (**P

    Journal: Scientific Reports

    Article Title: The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1

    doi: 10.1038/srep27597

    Figure Lengend Snippet: The overexpression of TRADD restores ORF3-inhibited NF-kB signaling. ( A ) TRADD plasmids were co-transfected with ORF3 at a ratio of 1:2 for 48 h, and the cells were then treated with Poly(I:C) for 12 h. Phosphorylated IκBα was assessed to measure NF-κB activity using western blotting, with β-actin serving as a loading control. Overexpression of TRADD restores ORF3-inhibited NF-κB signaling; the relative densitometric ratios for experimental bands are shown (**P

    Article Snippet: Antibodies specific for TLR3 (Catalogue # sc-32232, 1:300 dilution), TLR7 (# sc-57463, 1:300 dilution), TLR8 (# sc-373760, 1:300 dilution), TLR9 (# sc-52966, 1:400 dilution) and phospho-IκBα (# sc-8404, 1:400 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Over Expression, Transfection, Activity Assay, Western Blot

    ORF3 suppresses NF-κB signaling via TRADD and RIP1. ( A ) ORF3 suppresses the phosphorylation of IKKβ and IκBα. ( B ) TLR3 pulls down of key downstream molecules detected by a coimmunoprecipitation assay. ( C,D ) TRADD and TAK1 expression levels are down-regulated in ORF3-pretreated cells compared with GFP-pretreated cells after stimulation with Poly(I:C). In contrast, TRIF, TRAF6 and RIP1 expression levels are increased by this stimulation.

    Journal: Scientific Reports

    Article Title: The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1

    doi: 10.1038/srep27597

    Figure Lengend Snippet: ORF3 suppresses NF-κB signaling via TRADD and RIP1. ( A ) ORF3 suppresses the phosphorylation of IKKβ and IκBα. ( B ) TLR3 pulls down of key downstream molecules detected by a coimmunoprecipitation assay. ( C,D ) TRADD and TAK1 expression levels are down-regulated in ORF3-pretreated cells compared with GFP-pretreated cells after stimulation with Poly(I:C). In contrast, TRIF, TRAF6 and RIP1 expression levels are increased by this stimulation.

    Article Snippet: Antibodies specific for TLR3 (Catalogue # sc-32232, 1:300 dilution), TLR7 (# sc-57463, 1:300 dilution), TLR8 (# sc-373760, 1:300 dilution), TLR9 (# sc-52966, 1:400 dilution) and phospho-IκBα (# sc-8404, 1:400 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Co-Immunoprecipitation Assay, Expressing

    K377R mutation of RIP1 abolishes the TRADD-mediated restoration of NF-κB activation. ( A ) Pretreated cells were harvested for immunoprecipitation with an anti-RIP1 antibody to pull down K63-Ub. ORF3 inhibits K63-Ub of RIP1. ( B ) Overexpression of TRADD restores RIP1 K63-Ub. ( C ) K63-Ub is absent from K377R-mutated RIP1. (D) TRADD, RIP1 and mutant RIP1 plasmids were co-transfected with ORF3 at a ratio of 1:2 for 48 h, and the cells were then treat with Poly(I:C) for 12 h. The expression of phosphorylated IκBα was assessed to measure NF-κB activity using western blotting, with β-actin as a loading control. In the presence of Poly(I:C) and ORF3, K377R- mutated RIP1 decreased the level of phosphorylated IκBα, thus abolishing the NF-κB activation restored by TRADD (**P

    Journal: Scientific Reports

    Article Title: The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1

    doi: 10.1038/srep27597

    Figure Lengend Snippet: K377R mutation of RIP1 abolishes the TRADD-mediated restoration of NF-κB activation. ( A ) Pretreated cells were harvested for immunoprecipitation with an anti-RIP1 antibody to pull down K63-Ub. ORF3 inhibits K63-Ub of RIP1. ( B ) Overexpression of TRADD restores RIP1 K63-Ub. ( C ) K63-Ub is absent from K377R-mutated RIP1. (D) TRADD, RIP1 and mutant RIP1 plasmids were co-transfected with ORF3 at a ratio of 1:2 for 48 h, and the cells were then treat with Poly(I:C) for 12 h. The expression of phosphorylated IκBα was assessed to measure NF-κB activity using western blotting, with β-actin as a loading control. In the presence of Poly(I:C) and ORF3, K377R- mutated RIP1 decreased the level of phosphorylated IκBα, thus abolishing the NF-κB activation restored by TRADD (**P

    Article Snippet: Antibodies specific for TLR3 (Catalogue # sc-32232, 1:300 dilution), TLR7 (# sc-57463, 1:300 dilution), TLR8 (# sc-373760, 1:300 dilution), TLR9 (# sc-52966, 1:400 dilution) and phospho-IκBα (# sc-8404, 1:400 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Mutagenesis, Activation Assay, Immunoprecipitation, Over Expression, Transfection, Expressing, Activity Assay, Western Blot

    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

    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, 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