nf κ b  (Millipore)


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  • 99
    Name:
    ANTI PHOSPHO NF KAPPA B S536 antibody
    Description:

    Catalog Number:
    sab1305598
    Price:
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    Structured Review

    Millipore nf κ b
    <t>SIRT1</t> selectively attenuates transcription activity of NF- <t>κ</t> B and suppresses IL-12 expression in LPS-stimulated RAW 264.7 cells. (a) Cells were treated with 1 μ M STO-609 alone or combined with 2 μ M SRT1720 or 1 mM AICAR for 30 min before 100 ng/mL LPS stimulation (left). Cells were treated with 5 μ M Compound C alone or with 2 μ M SRT1720. Cells were also treated with 1 mM AICAR alone or with 2 μ M EX527 (right). Supernatants were collected at 24 h and IL-12 p40/IL-12 p70 (c) and IL-6 (d) levels were detected by ELISA ( n = 3). (b) Cells were transfected with plasmid pGL-luc2P/NF- κ BRE for 24 h and further treated with 100 ng/mL LPS in normal DMEM, calcium-free DMEM, DMEM with 2 mM CaCl 2 , DMEM with 1 mM AICAR, or DMEM with 2 μ M SRT1720 for 6 h. Relative luciferase activity of NF- κ B against medium was presented. ∗∗ P

    https://www.bioz.com/result/nf κ b/product/Millipore
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    Images

    1) Product Images from "Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK β-AMPK-SIRT1 Signaling Pathway"

    Article Title: Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK β-AMPK-SIRT1 Signaling Pathway

    Journal: Mediators of Inflammation

    doi: 10.1155/2016/6152713

    SIRT1 selectively attenuates transcription activity of NF- κ B and suppresses IL-12 expression in LPS-stimulated RAW 264.7 cells. (a) Cells were treated with 1 μ M STO-609 alone or combined with 2 μ M SRT1720 or 1 mM AICAR for 30 min before 100 ng/mL LPS stimulation (left). Cells were treated with 5 μ M Compound C alone or with 2 μ M SRT1720. Cells were also treated with 1 mM AICAR alone or with 2 μ M EX527 (right). Supernatants were collected at 24 h and IL-12 p40/IL-12 p70 (c) and IL-6 (d) levels were detected by ELISA ( n = 3). (b) Cells were transfected with plasmid pGL-luc2P/NF- κ BRE for 24 h and further treated with 100 ng/mL LPS in normal DMEM, calcium-free DMEM, DMEM with 2 mM CaCl 2 , DMEM with 1 mM AICAR, or DMEM with 2 μ M SRT1720 for 6 h. Relative luciferase activity of NF- κ B against medium was presented. ∗∗ P
    Figure Legend Snippet: SIRT1 selectively attenuates transcription activity of NF- κ B and suppresses IL-12 expression in LPS-stimulated RAW 264.7 cells. (a) Cells were treated with 1 μ M STO-609 alone or combined with 2 μ M SRT1720 or 1 mM AICAR for 30 min before 100 ng/mL LPS stimulation (left). Cells were treated with 5 μ M Compound C alone or with 2 μ M SRT1720. Cells were also treated with 1 mM AICAR alone or with 2 μ M EX527 (right). Supernatants were collected at 24 h and IL-12 p40/IL-12 p70 (c) and IL-6 (d) levels were detected by ELISA ( n = 3). (b) Cells were transfected with plasmid pGL-luc2P/NF- κ BRE for 24 h and further treated with 100 ng/mL LPS in normal DMEM, calcium-free DMEM, DMEM with 2 mM CaCl 2 , DMEM with 1 mM AICAR, or DMEM with 2 μ M SRT1720 for 6 h. Relative luciferase activity of NF- κ B against medium was presented. ∗∗ P

    Techniques Used: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Luciferase

    2) Product Images from "Down-Regulation of Myeloid Cell Leukemia 1 by Epigallocatechin-3-Gallate Sensitizes Rheumatoid Arthritis Synovial Fibroblasts to Tumor Necrosis Factor α-Induced Apoptosis"

    Article Title: Down-Regulation of Myeloid Cell Leukemia 1 by Epigallocatechin-3-Gallate Sensitizes Rheumatoid Arthritis Synovial Fibroblasts to Tumor Necrosis Factor α-Induced Apoptosis

    Journal: Arthritis and rheumatism

    doi: 10.1002/art.24488

    EGCG regulates Mcl-1 expression by inhibiting the NF- κ B and Akt pathways. A, Mcl-1 expression in RA synovial fibroblasts (2 × 10 5 /well) treated with pyrrolidine dithiocarbamate (PDTC; 100 μM ) alone, LY294002 (LY; 20 μM
    Figure Legend Snippet: EGCG regulates Mcl-1 expression by inhibiting the NF- κ B and Akt pathways. A, Mcl-1 expression in RA synovial fibroblasts (2 × 10 5 /well) treated with pyrrolidine dithiocarbamate (PDTC; 100 μM ) alone, LY294002 (LY; 20 μM

    Techniques Used: Expressing

    3) Product Images from "In Vivo Short-Term Topical Application of BAY 11-7082 Prevents the Acidic Bile–Induced mRNA and miRNA Oncogenic Phenotypes in Exposed Murine Hypopharyngeal Mucosa"

    Article Title: In Vivo Short-Term Topical Application of BAY 11-7082 Prevents the Acidic Bile–Induced mRNA and miRNA Oncogenic Phenotypes in Exposed Murine Hypopharyngeal Mucosa

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.02.001

    Topical application of BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in short-term 7-day–exposed murine HM. (A-a) IHC analysis for p-NF-κB (p65 S536) (from left to right): control untreated HM , revealing cytoplasmic staining; saline-treated HM , revealing sporadic and weak cytoplasmic or nuclear staining in a few basal cells; acidic bile–treated HM , producing intense nuclear and cytoplasmic staining of basal and parabasal/suprabasal layers; and acidic bile+inhibitor (BAY 11 7082)–treated HM , demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers. (b) Image analysis algorithm(s) ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm(s) illustrating mucosal and cellular compartments demonstrating p-NF- κ B staining]. (B) Acidic bile (pH 3.0) induces significantly higher positive nuclear p-NF- κ B (p65 S536) levels compared to saline-treated HM or untreated control. Topical application of BAY 11-7082 significantly decreases nuclear p-NF- κ B levels in acidic bile (pH 3.0) HM ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity=nuclear-positive/total-positive p-p65 staining). (C-a) IHC analysis for p-NF-κB (p65 S536) and (b) corresponding image analysis algorithms in murine tongue mucosa (used as internal control for analyzed HM in each group) (from left to right): control untreated TM, revealing weak cytoplasmic staining; saline-treated TM, acidic-bile-treated TM and acidic-bile+inhibitor (BAY 11 7082)–treated TM , producing sporadic weak cytoplasmic or nuclear staining in scattered basal cells ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining).
    Figure Legend Snippet: Topical application of BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in short-term 7-day–exposed murine HM. (A-a) IHC analysis for p-NF-κB (p65 S536) (from left to right): control untreated HM , revealing cytoplasmic staining; saline-treated HM , revealing sporadic and weak cytoplasmic or nuclear staining in a few basal cells; acidic bile–treated HM , producing intense nuclear and cytoplasmic staining of basal and parabasal/suprabasal layers; and acidic bile+inhibitor (BAY 11 7082)–treated HM , demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers. (b) Image analysis algorithm(s) ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm(s) illustrating mucosal and cellular compartments demonstrating p-NF- κ B staining]. (B) Acidic bile (pH 3.0) induces significantly higher positive nuclear p-NF- κ B (p65 S536) levels compared to saline-treated HM or untreated control. Topical application of BAY 11-7082 significantly decreases nuclear p-NF- κ B levels in acidic bile (pH 3.0) HM ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity=nuclear-positive/total-positive p-p65 staining). (C-a) IHC analysis for p-NF-κB (p65 S536) and (b) corresponding image analysis algorithms in murine tongue mucosa (used as internal control for analyzed HM in each group) (from left to right): control untreated TM, revealing weak cytoplasmic staining; saline-treated TM, acidic-bile-treated TM and acidic-bile+inhibitor (BAY 11 7082)–treated TM , producing sporadic weak cytoplasmic or nuclear staining in scattered basal cells ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining).

    Techniques Used: Activation Assay, Immunohistochemistry, Staining, Software, Generated

    In vivo short-term exposure to acidic bile, inducing transcriptional activation of NF- κ B related genes with antiapoptotic, cell proliferation, or oncogenic function, is prevented by topical application of BAY 11-7082. (A) Columns of graphs created by Graph Pad Prism software 6.0 depict mRNA phenotype in short-term exposed HM to (a) acidic bile (pH 3.0), (b) acidic bile (pH 3.0) + BAY 11-7082, and (c) saline (pH 7.0). mRNA phenotypes correspond to transcriptional expression ratios of NF- κ B–related genes bcl-2, RELA(p65), EGFR, WNT5A, STAT3, TNF-α , IL-6, and IL-1β comparing treated HM to corresponding mucosa from TM, used as internal control, by real-time qPCR analysis (mRNA levels of each target gene were normalized to GAPDH ). We observe an upregulation of mRNA expression of the analyzed genes in acidic bile–treated HM. BAY 11-7082 reverses the acidic bile–induced mRNA phenotype, demonstrating a phenotype similar to saline-induced mRNA. (B) Graphs, created by Graph Pad Prism 6 software, reveal transcriptional levels (normalized to GAPDH ) for the analyzed genes comparing HM exposed to acidic bile, acidic bile+BAY 11-7082, and saline to corresponding TM by real-time qPCR analysis ( P value
    Figure Legend Snippet: In vivo short-term exposure to acidic bile, inducing transcriptional activation of NF- κ B related genes with antiapoptotic, cell proliferation, or oncogenic function, is prevented by topical application of BAY 11-7082. (A) Columns of graphs created by Graph Pad Prism software 6.0 depict mRNA phenotype in short-term exposed HM to (a) acidic bile (pH 3.0), (b) acidic bile (pH 3.0) + BAY 11-7082, and (c) saline (pH 7.0). mRNA phenotypes correspond to transcriptional expression ratios of NF- κ B–related genes bcl-2, RELA(p65), EGFR, WNT5A, STAT3, TNF-α , IL-6, and IL-1β comparing treated HM to corresponding mucosa from TM, used as internal control, by real-time qPCR analysis (mRNA levels of each target gene were normalized to GAPDH ). We observe an upregulation of mRNA expression of the analyzed genes in acidic bile–treated HM. BAY 11-7082 reverses the acidic bile–induced mRNA phenotype, demonstrating a phenotype similar to saline-induced mRNA. (B) Graphs, created by Graph Pad Prism 6 software, reveal transcriptional levels (normalized to GAPDH ) for the analyzed genes comparing HM exposed to acidic bile, acidic bile+BAY 11-7082, and saline to corresponding TM by real-time qPCR analysis ( P value

    Techniques Used: In Vivo, Activation Assay, Software, Expressing, Real-time Polymerase Chain Reaction

    BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in MHPC. (A) Western blot analysis was performed in nuclear and cytoplasmic protein extracts of treated MHPC with and without BAY 11-7082, demonstrating that NF- κ B inhibitor induced a significant reduction of activated NF- κ B. Graphs depict significantly lower (a) nuclear p-NF- κ B (p65 S536) and (b) cytoplasmic p-I κ B-α (Ser32/36) levels in acidic bile–treated groups (pH 4.0) ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (Nuclear p-NF-κB protein levels were normalized to Histone 1; cytoplasmic p-IκB-α and bcl-2 protein levels were normalized to β-actin). (B) Graphs created by Graph Pad Prism 6.0 software reveal ranks of BAY 11-7082–induced (a) nuclear p-NF-κB(p65 S536) and (b) cytoplasmic p-IκB-α (Ser32/36) protein ratios (with/without NF-κB inhibitor) between experimental and control treated groups. Acidic bile (pH 4.0)–treated MHPC demonstrates the most significant reduction of activated NF- κ B and p-IκB-α protein levels in the presence of ΒΑΥ 11-7082 ( P
    Figure Legend Snippet: BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in MHPC. (A) Western blot analysis was performed in nuclear and cytoplasmic protein extracts of treated MHPC with and without BAY 11-7082, demonstrating that NF- κ B inhibitor induced a significant reduction of activated NF- κ B. Graphs depict significantly lower (a) nuclear p-NF- κ B (p65 S536) and (b) cytoplasmic p-I κ B-α (Ser32/36) levels in acidic bile–treated groups (pH 4.0) ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (Nuclear p-NF-κB protein levels were normalized to Histone 1; cytoplasmic p-IκB-α and bcl-2 protein levels were normalized to β-actin). (B) Graphs created by Graph Pad Prism 6.0 software reveal ranks of BAY 11-7082–induced (a) nuclear p-NF-κB(p65 S536) and (b) cytoplasmic p-IκB-α (Ser32/36) protein ratios (with/without NF-κB inhibitor) between experimental and control treated groups. Acidic bile (pH 4.0)–treated MHPC demonstrates the most significant reduction of activated NF- κ B and p-IκB-α protein levels in the presence of ΒΑΥ 11-7082 ( P

    Techniques Used: Activation Assay, Western Blot, Software

    Short-term exposure of murine HM to acidic bile upregulates the NF- κ B signaling pathway and is reduced by topical application of BAY 11-7082. (A). Table presents the expression changes (upregulation or downregulation by two-fold changes) of 84 genes of NF- κ B signaling pathway, analyzed by PCR microarray analysis, in acidic bile–treated HM under the topical application of BAY 11-7082. (B) Heat maps were obtained by RT 2 -Profiler PCR array analysis for NF- κ B signaling. Genes were clustered based on their biological role. Heat maps demonstrated the effect of BAY 11-7082 in gene expression of NF- κ B pathway and NF- κ B responsive genes (red color for maximum expression and green for minimum). Group 1: acidic bile–treated HM; control group: saline treated-HM, and group 2: acidic bile with BAY 11-7082–treated HM [gene expression has been normalized to two housekeeping genes: GAPDH, glyceraldehyde-3-phosphate dehydrogenase and Hsp90ab1, Heat shock protein 90 alpha (cytosolic), class B member 1].
    Figure Legend Snippet: Short-term exposure of murine HM to acidic bile upregulates the NF- κ B signaling pathway and is reduced by topical application of BAY 11-7082. (A). Table presents the expression changes (upregulation or downregulation by two-fold changes) of 84 genes of NF- κ B signaling pathway, analyzed by PCR microarray analysis, in acidic bile–treated HM under the topical application of BAY 11-7082. (B) Heat maps were obtained by RT 2 -Profiler PCR array analysis for NF- κ B signaling. Genes were clustered based on their biological role. Heat maps demonstrated the effect of BAY 11-7082 in gene expression of NF- κ B pathway and NF- κ B responsive genes (red color for maximum expression and green for minimum). Group 1: acidic bile–treated HM; control group: saline treated-HM, and group 2: acidic bile with BAY 11-7082–treated HM [gene expression has been normalized to two housekeeping genes: GAPDH, glyceraldehyde-3-phosphate dehydrogenase and Hsp90ab1, Heat shock protein 90 alpha (cytosolic), class B member 1].

    Techniques Used: Expressing, Polymerase Chain Reaction, Microarray

    4) Product Images from "Cytokine Effects on Cell Viability and Death of Prostate Carcinoma Cells"

    Article Title: Cytokine Effects on Cell Viability and Death of Prostate Carcinoma Cells

    Journal: BioMed Research International

    doi: 10.1155/2014/536049

    Western blot analysis of LNCaP protein expression. (a) and (b) LNCaP cells were treated with TNF- α (10 and 100 ng/mL) for 24 h or IFN- γ (25 ng/mL) for 30, 90 min, and 24 h or pretreated with LY-29004 (20 μ M) for 1 h and then treated with IFN- γ (25 ng/mL) for 90 min. Untreated cells for 90 min and 24 h were used as controls (ctrl). (c) and (d) LNCaP cells were treated with IL-1 β (5 ng/mL) for 30, 90 min, and 24 h or pretreated with UO126 (30 μ M), BAY-117082 (30 μ M), or SP600125 (30 μ M) for 1 h and then treated with IL-1 β (5 ng/mL) for 90 min. Untreated cells for 90 min and 24 h were used as controls (ctrl). (e) and (f) LNCaP and PC-3 cells were treated with IL-1 β (5 ng/mL) or TNF- α (10 and 100 ng/mL) or SP600125 (30 μ M) or UO126 (30 μ M) for 72 h, or pretreated with SP600125 (30 μ M) or UO126 (30 μ M) for 1 h and then treated with IL-1 β (5 ng/mL) for 72 h. Untreated cells for 72 h were used as controls (ctrl). Cell extracts were resolved by SDS-PAGE and analyzed by western blot with antibodies against Bid, Bad, Bax, Bcl-2, Cyclin D1, p-ERK 1/2, p-p38, p-Akt, I κ B- α , c-IAP2, and caspase 3. Reprobing with antibody against α -tubulin or β -actin was used as a loading and transfer marker.
    Figure Legend Snippet: Western blot analysis of LNCaP protein expression. (a) and (b) LNCaP cells were treated with TNF- α (10 and 100 ng/mL) for 24 h or IFN- γ (25 ng/mL) for 30, 90 min, and 24 h or pretreated with LY-29004 (20 μ M) for 1 h and then treated with IFN- γ (25 ng/mL) for 90 min. Untreated cells for 90 min and 24 h were used as controls (ctrl). (c) and (d) LNCaP cells were treated with IL-1 β (5 ng/mL) for 30, 90 min, and 24 h or pretreated with UO126 (30 μ M), BAY-117082 (30 μ M), or SP600125 (30 μ M) for 1 h and then treated with IL-1 β (5 ng/mL) for 90 min. Untreated cells for 90 min and 24 h were used as controls (ctrl). (e) and (f) LNCaP and PC-3 cells were treated with IL-1 β (5 ng/mL) or TNF- α (10 and 100 ng/mL) or SP600125 (30 μ M) or UO126 (30 μ M) for 72 h, or pretreated with SP600125 (30 μ M) or UO126 (30 μ M) for 1 h and then treated with IL-1 β (5 ng/mL) for 72 h. Untreated cells for 72 h were used as controls (ctrl). Cell extracts were resolved by SDS-PAGE and analyzed by western blot with antibodies against Bid, Bad, Bax, Bcl-2, Cyclin D1, p-ERK 1/2, p-p38, p-Akt, I κ B- α , c-IAP2, and caspase 3. Reprobing with antibody against α -tubulin or β -actin was used as a loading and transfer marker.

    Techniques Used: Western Blot, Expressing, SDS Page, Marker

    Western blot analysis of LNCaP protein expression. (a), (b), (c), and (d) LNCaP cells were treated with TNF- α (10 and 100 ng/mL) for 24 h or IL-13 (20 and 100 ng/mL) for 30 and 90 min and 24 h or LY-29004 (20 μ M) for 90 min or pretreated with LY-29004 (20 μ M), BAY-117082 (30 μ M), or SP600125 (30 μ M) for 1 h and then treated with IL-13 (20 and 100 ng/mL) for 90 min. Untreated cells for 30 and 90 min and 24 h were used as control (ctrl). Cell extracts were resolved by SDS-PAGE and analyzed by western blot with antibodies against Bid, Bad, Bax, Bcl-2, Cyclin D1, p-ERK 1/2, I κ B- α , c-IAP2, p-p38, p-Akt, Fas, FLIP L , p-JNK, and Bcl-X L . Reprobing with antibody against α -tubulin or β -actin was used as a loading and transfer marker.
    Figure Legend Snippet: Western blot analysis of LNCaP protein expression. (a), (b), (c), and (d) LNCaP cells were treated with TNF- α (10 and 100 ng/mL) for 24 h or IL-13 (20 and 100 ng/mL) for 30 and 90 min and 24 h or LY-29004 (20 μ M) for 90 min or pretreated with LY-29004 (20 μ M), BAY-117082 (30 μ M), or SP600125 (30 μ M) for 1 h and then treated with IL-13 (20 and 100 ng/mL) for 90 min. Untreated cells for 30 and 90 min and 24 h were used as control (ctrl). Cell extracts were resolved by SDS-PAGE and analyzed by western blot with antibodies against Bid, Bad, Bax, Bcl-2, Cyclin D1, p-ERK 1/2, I κ B- α , c-IAP2, p-p38, p-Akt, Fas, FLIP L , p-JNK, and Bcl-X L . Reprobing with antibody against α -tubulin or β -actin was used as a loading and transfer marker.

    Techniques Used: Western Blot, Expressing, SDS Page, Marker

    5) Product Images from "AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIA-PaCa-2"

    Article Title: AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIA-PaCa-2

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601037

    Examination of the NF- κ B site at −736 nt within the BCL-2 promoter by EMSA. MIA-PaCa-2 nuclear lysate was incubated with radiolabelled oligonucleotides corresponding to the NF- κ B site (except lane 1) and underwent cold competition (100 × molar excess) of nucleotides corresponding to the NF- κ B site (lane 3), the site-directed mutant of the NF- κ B site (lane 4), or an unrelated transcriptional site, AP-1 (lane 5). Supershift of the NF- κ B complex was achieved using an antibody to the p50 subunit of NF- κ B (lane 6). Treatment of the cells with LY294002 eliminated the upper p50/p65 band of the typical NF- κ B gelshift pattern (lane 8).
    Figure Legend Snippet: Examination of the NF- κ B site at −736 nt within the BCL-2 promoter by EMSA. MIA-PaCa-2 nuclear lysate was incubated with radiolabelled oligonucleotides corresponding to the NF- κ B site (except lane 1) and underwent cold competition (100 × molar excess) of nucleotides corresponding to the NF- κ B site (lane 3), the site-directed mutant of the NF- κ B site (lane 4), or an unrelated transcriptional site, AP-1 (lane 5). Supershift of the NF- κ B complex was achieved using an antibody to the p50 subunit of NF- κ B (lane 6). Treatment of the cells with LY294002 eliminated the upper p50/p65 band of the typical NF- κ B gelshift pattern (lane 8).

    Techniques Used: Incubation, Mutagenesis

    6) Product Images from "In Vivo Short-Term Topical Application of BAY 11-7082 Prevents the Acidic Bile–Induced mRNA and miRNA Oncogenic Phenotypes in Exposed Murine Hypopharyngeal Mucosa"

    Article Title: In Vivo Short-Term Topical Application of BAY 11-7082 Prevents the Acidic Bile–Induced mRNA and miRNA Oncogenic Phenotypes in Exposed Murine Hypopharyngeal Mucosa

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2018.02.001

    Topical application of BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in short-term 7-day–exposed murine HM. (A-a) IHC analysis for p-NF-κB (p65 S536) (from left to right): control untreated HM , revealing cytoplasmic staining; saline-treated HM , revealing sporadic and weak cytoplasmic or nuclear staining in a few basal cells; acidic bile–treated HM , producing intense nuclear and cytoplasmic staining of basal and parabasal/suprabasal layers; and acidic bile+inhibitor (BAY 11 7082)–treated HM , demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers. (b) Image analysis algorithm(s) ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm(s) illustrating mucosal and cellular compartments demonstrating p-NF- κ B staining]. (B) Acidic bile (pH 3.0) induces significantly higher positive nuclear p-NF- κ B (p65 S536) levels compared to saline-treated HM or untreated control. Topical application of BAY 11-7082 significantly decreases nuclear p-NF- κ B levels in acidic bile (pH 3.0) HM ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity=nuclear-positive/total-positive p-p65 staining). (C-a) IHC analysis for p-NF-κB (p65 S536) and (b) corresponding image analysis algorithms in murine tongue mucosa (used as internal control for analyzed HM in each group) (from left to right): control untreated TM, revealing weak cytoplasmic staining; saline-treated TM, acidic-bile-treated TM and acidic-bile+inhibitor (BAY 11 7082)–treated TM , producing sporadic weak cytoplasmic or nuclear staining in scattered basal cells ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining).
    Figure Legend Snippet: Topical application of BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in short-term 7-day–exposed murine HM. (A-a) IHC analysis for p-NF-κB (p65 S536) (from left to right): control untreated HM , revealing cytoplasmic staining; saline-treated HM , revealing sporadic and weak cytoplasmic or nuclear staining in a few basal cells; acidic bile–treated HM , producing intense nuclear and cytoplasmic staining of basal and parabasal/suprabasal layers; and acidic bile+inhibitor (BAY 11 7082)–treated HM , demonstrating nuclear or cytoplasmic staining mainly of cells of basal layer and weak cytoplasmic staining of suprabasal layers. (b) Image analysis algorithm(s) ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining) [Images were captured using Aperio CS2 and analyzed by Image Scope software (Leica Microsystems, Buffalo Grove, IL) that generated algorithm(s) illustrating mucosal and cellular compartments demonstrating p-NF- κ B staining]. (B) Acidic bile (pH 3.0) induces significantly higher positive nuclear p-NF- κ B (p65 S536) levels compared to saline-treated HM or untreated control. Topical application of BAY 11-7082 significantly decreases nuclear p-NF- κ B levels in acidic bile (pH 3.0) HM ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (positivity=nuclear-positive/total-positive p-p65 staining). (C-a) IHC analysis for p-NF-κB (p65 S536) and (b) corresponding image analysis algorithms in murine tongue mucosa (used as internal control for analyzed HM in each group) (from left to right): control untreated TM, revealing weak cytoplasmic staining; saline-treated TM, acidic-bile-treated TM and acidic-bile+inhibitor (BAY 11 7082)–treated TM , producing sporadic weak cytoplasmic or nuclear staining in scattered basal cells ( red : positive nuclear staining of p-NF- κ B; orange : intense positive cytoplasmic staining of p-NF- κ B; yellow : weak cytoplasmic staining of p-NF- κ B; blue : negative p-NF- κ B staining).

    Techniques Used: Activation Assay, Immunohistochemistry, Staining, Software, Generated

    In vivo short-term exposure to acidic bile, inducing transcriptional activation of NF- κ B related genes with antiapoptotic, cell proliferation, or oncogenic function, is prevented by topical application of BAY 11-7082. (A) Columns of graphs created by Graph Pad Prism software 6.0 depict mRNA phenotype in short-term exposed HM to (a) acidic bile (pH 3.0), (b) acidic bile (pH 3.0) + BAY 11-7082, and (c) saline (pH 7.0). mRNA phenotypes correspond to transcriptional expression ratios of NF- κ B–related genes bcl-2, RELA(p65), EGFR, WNT5A, STAT3, TNF-α , IL-6, and IL-1β comparing treated HM to corresponding mucosa from TM, used as internal control, by real-time qPCR analysis (mRNA levels of each target gene were normalized to GAPDH ). We observe an upregulation of mRNA expression of the analyzed genes in acidic bile–treated HM. BAY 11-7082 reverses the acidic bile–induced mRNA phenotype, demonstrating a phenotype similar to saline-induced mRNA. (B) Graphs, created by Graph Pad Prism 6 software, reveal transcriptional levels (normalized to GAPDH ) for the analyzed genes comparing HM exposed to acidic bile, acidic bile+BAY 11-7082, and saline to corresponding TM by real-time qPCR analysis ( P value
    Figure Legend Snippet: In vivo short-term exposure to acidic bile, inducing transcriptional activation of NF- κ B related genes with antiapoptotic, cell proliferation, or oncogenic function, is prevented by topical application of BAY 11-7082. (A) Columns of graphs created by Graph Pad Prism software 6.0 depict mRNA phenotype in short-term exposed HM to (a) acidic bile (pH 3.0), (b) acidic bile (pH 3.0) + BAY 11-7082, and (c) saline (pH 7.0). mRNA phenotypes correspond to transcriptional expression ratios of NF- κ B–related genes bcl-2, RELA(p65), EGFR, WNT5A, STAT3, TNF-α , IL-6, and IL-1β comparing treated HM to corresponding mucosa from TM, used as internal control, by real-time qPCR analysis (mRNA levels of each target gene were normalized to GAPDH ). We observe an upregulation of mRNA expression of the analyzed genes in acidic bile–treated HM. BAY 11-7082 reverses the acidic bile–induced mRNA phenotype, demonstrating a phenotype similar to saline-induced mRNA. (B) Graphs, created by Graph Pad Prism 6 software, reveal transcriptional levels (normalized to GAPDH ) for the analyzed genes comparing HM exposed to acidic bile, acidic bile+BAY 11-7082, and saline to corresponding TM by real-time qPCR analysis ( P value

    Techniques Used: In Vivo, Activation Assay, Software, Expressing, Real-time Polymerase Chain Reaction

    BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in MHPC. (A) Western blot analysis was performed in nuclear and cytoplasmic protein extracts of treated MHPC with and without BAY 11-7082, demonstrating that NF- κ B inhibitor induced a significant reduction of activated NF- κ B. Graphs depict significantly lower (a) nuclear p-NF- κ B (p65 S536) and (b) cytoplasmic p-I κ B-α (Ser32/36) levels in acidic bile–treated groups (pH 4.0) ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (Nuclear p-NF-κB protein levels were normalized to Histone 1; cytoplasmic p-IκB-α and bcl-2 protein levels were normalized to β-actin). (B) Graphs created by Graph Pad Prism 6.0 software reveal ranks of BAY 11-7082–induced (a) nuclear p-NF-κB(p65 S536) and (b) cytoplasmic p-IκB-α (Ser32/36) protein ratios (with/without NF-κB inhibitor) between experimental and control treated groups. Acidic bile (pH 4.0)–treated MHPC demonstrates the most significant reduction of activated NF- κ B and p-IκB-α protein levels in the presence of ΒΑΥ 11-7082 ( P
    Figure Legend Snippet: BAY 11-7082 inhibits the acidic bile–induced NF- κ B activation in MHPC. (A) Western blot analysis was performed in nuclear and cytoplasmic protein extracts of treated MHPC with and without BAY 11-7082, demonstrating that NF- κ B inhibitor induced a significant reduction of activated NF- κ B. Graphs depict significantly lower (a) nuclear p-NF- κ B (p65 S536) and (b) cytoplasmic p-I κ B-α (Ser32/36) levels in acidic bile–treated groups (pH 4.0) ( P values by t test; multiple comparisons by Holm-Sidak; GraphPad Prism 6.0) (Nuclear p-NF-κB protein levels were normalized to Histone 1; cytoplasmic p-IκB-α and bcl-2 protein levels were normalized to β-actin). (B) Graphs created by Graph Pad Prism 6.0 software reveal ranks of BAY 11-7082–induced (a) nuclear p-NF-κB(p65 S536) and (b) cytoplasmic p-IκB-α (Ser32/36) protein ratios (with/without NF-κB inhibitor) between experimental and control treated groups. Acidic bile (pH 4.0)–treated MHPC demonstrates the most significant reduction of activated NF- κ B and p-IκB-α protein levels in the presence of ΒΑΥ 11-7082 ( P

    Techniques Used: Activation Assay, Western Blot, Software

    Short-term exposure of murine HM to acidic bile upregulates the NF- κ B signaling pathway and is reduced by topical application of BAY 11-7082. (A). Table presents the expression changes (upregulation or downregulation by two-fold changes) of 84 genes of NF- κ B signaling pathway, analyzed by PCR microarray analysis, in acidic bile–treated HM under the topical application of BAY 11-7082. (B) Heat maps were obtained by RT 2 -Profiler PCR array analysis for NF- κ B signaling. Genes were clustered based on their biological role. Heat maps demonstrated the effect of BAY 11-7082 in gene expression of NF- κ B pathway and NF- κ B responsive genes (red color for maximum expression and green for minimum). Group 1: acidic bile–treated HM; control group: saline treated-HM, and group 2: acidic bile with BAY 11-7082–treated HM [gene expression has been normalized to two housekeeping genes: GAPDH, glyceraldehyde-3-phosphate dehydrogenase and Hsp90ab1, Heat shock protein 90 alpha (cytosolic), class B member 1].
    Figure Legend Snippet: Short-term exposure of murine HM to acidic bile upregulates the NF- κ B signaling pathway and is reduced by topical application of BAY 11-7082. (A). Table presents the expression changes (upregulation or downregulation by two-fold changes) of 84 genes of NF- κ B signaling pathway, analyzed by PCR microarray analysis, in acidic bile–treated HM under the topical application of BAY 11-7082. (B) Heat maps were obtained by RT 2 -Profiler PCR array analysis for NF- κ B signaling. Genes were clustered based on their biological role. Heat maps demonstrated the effect of BAY 11-7082 in gene expression of NF- κ B pathway and NF- κ B responsive genes (red color for maximum expression and green for minimum). Group 1: acidic bile–treated HM; control group: saline treated-HM, and group 2: acidic bile with BAY 11-7082–treated HM [gene expression has been normalized to two housekeeping genes: GAPDH, glyceraldehyde-3-phosphate dehydrogenase and Hsp90ab1, Heat shock protein 90 alpha (cytosolic), class B member 1].

    Techniques Used: Expressing, Polymerase Chain Reaction, Microarray

    7) Product Images from "MicroRNA-21 Promotes Proliferation of Fibroblast-Like Synoviocytes through Mediation of NF-κB Nuclear Translocation in a Rat Model of Collagen-Induced Rheumatoid Arthritis"

    Article Title: MicroRNA-21 Promotes Proliferation of Fibroblast-Like Synoviocytes through Mediation of NF-κB Nuclear Translocation in a Rat Model of Collagen-Induced Rheumatoid Arthritis

    Journal: BioMed Research International

    doi: 10.1155/2016/9279078

    The effects of overexpressed miR-21 on nucleoprotein NF- κ B level and cell viability. (a) The miR-21 levels in normal FLS after pro-miR-21 treatment. (b) The determination of nucleoprotein NF- κ B levels using western blot methods. (c) The change of normal FLS proliferation rate after pro-miR-21 treatment. (d) the nucleoprotein NF- κ B level detection after additional BAY 11-7082 treatment in normal FLS. (e) The change in FLS proliferation rate after the additional BAY 11-7082 treatment in normal FLS. ∗ P
    Figure Legend Snippet: The effects of overexpressed miR-21 on nucleoprotein NF- κ B level and cell viability. (a) The miR-21 levels in normal FLS after pro-miR-21 treatment. (b) The determination of nucleoprotein NF- κ B levels using western blot methods. (c) The change of normal FLS proliferation rate after pro-miR-21 treatment. (d) the nucleoprotein NF- κ B level detection after additional BAY 11-7082 treatment in normal FLS. (e) The change in FLS proliferation rate after the additional BAY 11-7082 treatment in normal FLS. ∗ P

    Techniques Used: Western Blot

    Evaluation of miR-21 and nucleoprotein NF- κ B levels in the RA group and normal control. (a) NF- κ B expression pattern in the RA group and normal control. (b) The miR-21 level was detected by Q-PCR in the RA group and normal control. N indicates the normal control. RA represents the RA group. ∗ P
    Figure Legend Snippet: Evaluation of miR-21 and nucleoprotein NF- κ B levels in the RA group and normal control. (a) NF- κ B expression pattern in the RA group and normal control. (b) The miR-21 level was detected by Q-PCR in the RA group and normal control. N indicates the normal control. RA represents the RA group. ∗ P

    Techniques Used: Expressing, Polymerase Chain Reaction

    The effects of downregulated miR-21 on nucleoprotein NF- κ B level and cell viability. (a) The miR-21 levels in RA FLS after anti-miR-21 treatment. (b) The determination of nucleoprotein NF- κ B levels using western blotting methods. (c) The change in the RA FLS proliferation rate after anti-miR-21 treatment. (d) The nucleoprotein NF- κ B level after BAY 11-7082 treatment in RA FLS. (e) The change in the FLS proliferation rate after BAY 11-7082 treatment in RA FLS. ∗ P
    Figure Legend Snippet: The effects of downregulated miR-21 on nucleoprotein NF- κ B level and cell viability. (a) The miR-21 levels in RA FLS after anti-miR-21 treatment. (b) The determination of nucleoprotein NF- κ B levels using western blotting methods. (c) The change in the RA FLS proliferation rate after anti-miR-21 treatment. (d) The nucleoprotein NF- κ B level after BAY 11-7082 treatment in RA FLS. (e) The change in the FLS proliferation rate after BAY 11-7082 treatment in RA FLS. ∗ P

    Techniques Used: Western Blot

    8) Product Images from "Tumor necrosis factor α decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription. Tumor necrosis factor α decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription"

    Article Title: Tumor necrosis factor α decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription. Tumor necrosis factor α decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription

    Journal: Physiological Reports

    doi: 10.14814/phy2.13451

    AQP 3 mRNA and protein expression are decreased following treatment with TNF α and this effect can be prevented by pretreatment with inhibitors of MEK / ERK and NF ‐ κ B signaling pathways. AQP 3 mRNA expression was assessed by real‐time RT ‐ PCR in HT ‐29 cells treated with TNF α (25 ng/mL) for the times indicated (A) or for a total of 12 h in a concentration‐dependent fashion as indicated (B). Statistical significance was assessed in comparison to untreated samples using an unpaired t ‐test (A) or ANOVA (B) (* P
    Figure Legend Snippet: AQP 3 mRNA and protein expression are decreased following treatment with TNF α and this effect can be prevented by pretreatment with inhibitors of MEK / ERK and NF ‐ κ B signaling pathways. AQP 3 mRNA expression was assessed by real‐time RT ‐ PCR in HT ‐29 cells treated with TNF α (25 ng/mL) for the times indicated (A) or for a total of 12 h in a concentration‐dependent fashion as indicated (B). Statistical significance was assessed in comparison to untreated samples using an unpaired t ‐test (A) or ANOVA (B) (* P

    Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay

    9) Product Images from "Curcumin Analogue CA15 Exhibits Anticancer Effects on HEp-2 Cells via Targeting NF-κB"

    Article Title: Curcumin Analogue CA15 Exhibits Anticancer Effects on HEp-2 Cells via Targeting NF-κB

    Journal: BioMed Research International

    doi: 10.1155/2017/4751260

    CA15 inhibited TNF- α -induced NF- κ B activation in HEp-2 cells. (a and b) HEp-2 cells were pretreated with different concentrations (0–20 μ M) of CA15 and 20 μ M BMS for 1 h followed by incubation with TNF- α (5 ng/ml) for 15 min (a) or 30 min (b). The protein levels of p-IKK, IKK, and I κ B were examined by Western blot. The graphs display means ± SEM of 3 independent experiments. ∗∗ P
    Figure Legend Snippet: CA15 inhibited TNF- α -induced NF- κ B activation in HEp-2 cells. (a and b) HEp-2 cells were pretreated with different concentrations (0–20 μ M) of CA15 and 20 μ M BMS for 1 h followed by incubation with TNF- α (5 ng/ml) for 15 min (a) or 30 min (b). The protein levels of p-IKK, IKK, and I κ B were examined by Western blot. The graphs display means ± SEM of 3 independent experiments. ∗∗ P

    Techniques Used: Activation Assay, Incubation, Western Blot

    10) Product Images from "Protective Effects and Possible Mechanisms of Ergothioneine and Hispidin against Methylglyoxal-Induced Injuries in Rat Pheochromocytoma Cells"

    Article Title: Protective Effects and Possible Mechanisms of Ergothioneine and Hispidin against Methylglyoxal-Induced Injuries in Rat Pheochromocytoma Cells

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/4824371

    Effects of ergothioneine (EGT), hispidin (HIP), and EGT + HIP on cytoplasm I κ B (Cyt-I κ B), cytoplasm NF- κ B (Cyt-NF- κ B), and nuclear NF- κ B (Nu-NF κ B) protein expression in PC12 cells treated with 30 mM glucose and 30 μ M methylglyoxal GLU + MGO) or 30 mM mannitol (to exclude the impact of glucose osmosis). β -Actin serves as an internal control. PC12 cells were pretreated with epalrestat (EPA), aminoguanidine (AMG), ETG, HIP, and EGT + HIP for 2 h and incubated with GLU + MGO for 72 h. A: control; B: GLU + MGO; C: 30 mM mannitol; D: 10 μ M EPA; E: 100 nM AMG; F: 2 μ M EGT; G: 2 μ M HIP; H: 2 μ M EGT + 2 μ M HIP. (b–d) Quantitative data for Cyt-I κ B, Cyt-NF- κ B, and Nu-NF- κ B expression. A portion of the protein (50 μ g) was loaded to 10% SDS-PAGE. Values (means ± SD of triplicate tests) without a superscript letter are significantly different ( P
    Figure Legend Snippet: Effects of ergothioneine (EGT), hispidin (HIP), and EGT + HIP on cytoplasm I κ B (Cyt-I κ B), cytoplasm NF- κ B (Cyt-NF- κ B), and nuclear NF- κ B (Nu-NF κ B) protein expression in PC12 cells treated with 30 mM glucose and 30 μ M methylglyoxal GLU + MGO) or 30 mM mannitol (to exclude the impact of glucose osmosis). β -Actin serves as an internal control. PC12 cells were pretreated with epalrestat (EPA), aminoguanidine (AMG), ETG, HIP, and EGT + HIP for 2 h and incubated with GLU + MGO for 72 h. A: control; B: GLU + MGO; C: 30 mM mannitol; D: 10 μ M EPA; E: 100 nM AMG; F: 2 μ M EGT; G: 2 μ M HIP; H: 2 μ M EGT + 2 μ M HIP. (b–d) Quantitative data for Cyt-I κ B, Cyt-NF- κ B, and Nu-NF- κ B expression. A portion of the protein (50 μ g) was loaded to 10% SDS-PAGE. Values (means ± SD of triplicate tests) without a superscript letter are significantly different ( P

    Techniques Used: Expressing, Incubation, SDS Page

    11) Product Images from ""

    Article Title:

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.114.093039

    Involvement of p38 MAPK/NF- κ B pathway in the suppression of ethanol-induced Nox2 and p22 phox expression by gAcrp in RAW 264.7 macrophages. (A, B) Cells were pretreated with Bay 11-7082 (5 μ M), a selective inhibitor of IKK, for 1 hour, followed by stimulation with 100 mM ethanol for 24 hours. Messenger RNA levels of Nox2 (A) and p22 phox (B) were assessed by qRT-PCR as described previously. Values are expressed as mean ± S.E.M. ( n = 8). * P
    Figure Legend Snippet: Involvement of p38 MAPK/NF- κ B pathway in the suppression of ethanol-induced Nox2 and p22 phox expression by gAcrp in RAW 264.7 macrophages. (A, B) Cells were pretreated with Bay 11-7082 (5 μ M), a selective inhibitor of IKK, for 1 hour, followed by stimulation with 100 mM ethanol for 24 hours. Messenger RNA levels of Nox2 (A) and p22 phox (B) were assessed by qRT-PCR as described previously. Values are expressed as mean ± S.E.M. ( n = 8). * P

    Techniques Used: Expressing, Quantitative RT-PCR

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    Western Blot:

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    Article Snippet: .. The antibodies used for western blotting were anti-β-actin (1∶500; Abcam), anti-dentin sialoprotein (DSP, 1∶200; Santa Cruz biotechnology), anti-DMP1 (1∶200; Santa Cruz biotechnology), anti-NF (1∶1000; Millipore), anti-β-Tubulin III (1∶1000; Millipore), anti-ALP (1∶500; Abcam), anti-BSP (1∶100; Abcam), anti-OPN (1∶1000; Abcam), anti-COL1 (1∶500; Abcam), anti-periostin (1∶500; Abcam) and anti-TGFβ1 (1∶400; Abcam). .. In vivo studies on the odontogenic differentiation of DPCs and DFCs DFCs and DPCs (5×104 ) were seeded on TDM and cultured in vitro for three days during which the cells adhered and proliferated on the TDM and covered the dentinal tubules before they were implanted into the dorsum of nude mice (BALB/c nude mice ).

    Incubation:

    Article Title: Insulin-like growth factor 1 modulates bioengineered tooth morphogenesis
    Article Snippet: .. After sections were blocked with 1% goat serum (Vector Laboratories) in PBS for 1 h at room temperature, they were incubated with anti-NF (1:200; Chemicon, Temecula, CA, USA) or anti-VWF (1:1000; Dako, Glostrup, Denmark) antibodies for 2 h at room temperature. .. After sections were washed with 0.1% Tween 20 in PBS, they were treated with goat anti-rabbit IgG Alexa 488 (1:1000; Invitrogen) for 1 h at room temperature.

    Staining:

    Article Title: Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis
    Article Snippet: .. Cells were stained with anti-NF (EMD Millipore, Billerica, MA, USA), anti-MAP2 (EMD Millipore), anti-βIII-tubulin (Covance, Denver, PA, USA) and anti-cleaved casp3 (Cell signaling) antibodies. ..

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    Millipore nf κ b activation inhibitor ii jsh 23
    3-Cl-AHPC-mediated phosphorylation of IKK α and IKK β , and activation of the NF- <t>κ</t> B canonical and noncanonical pathways. ( a ) 3-Cl-AHPC activates NF- κ B in KG-1 cells; cells were transduced with lentiviral NF- κ B reporter (GFP) particles transiently for 48 h and then treated with 1 μ M 3-Cl-AHPC for 24 h. ( b ) NF- κ B activation inhibitor <t>JSH-23</t> blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μ M 3-Cl-AHPC for 24 h. ( c ) 3-Cl-AHPC enhances phosphorylation of IKK α and IKK β . ( d ) 3-Cl-AHPC induces phosphorylation of NF- κ Bp65/RelA at Ser276. ( e ) 3-Cl-AHPC enhances phosphorylation of NF- κ B2p100 followed by processing to p52 in both cell lines. ( f ) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF- κ B2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μ M) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. * and ** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively ( P -value is
    Nf κ B Activation Inhibitor Ii Jsh 23, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nf κ b activation
    Reducing basal NF- <t>κ</t> B activity upregulates Hgf expression in <t>MDSCs.</t> ( a ) Real-time RT-PCR analysis revealed that Hgf was significantly upregulated in p65 +/− compared with WT MDSCs (* P
    Nf κ B Activation, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore nf κ b p50 p65 ez tfa transcription factor assay kit
    Effect of TFRG on NF-κB, MAPK activities, and microRNA-155 expression in LPS/IFNγ-stimulated RAW264.7 cells . (a, b) DNA binding activity of <t>p50</t> and <t>p65</t> proteins in nuclear extracts was assessed using NF- κ Bp50/p65 <t>EZ-TFA</t> transcription factor assay. Absorbance was measured at 450 nm in a microplate spectrophotometer. Results were normalized to absorbance/mg protein. (c) RAW264.7 cells were plated at a density of 1 × 10 6 cells/well in 30-mm dish overnight. TFRG were added to cells for 1 h followed by 30-min stimulation of IFN γ (10 U/ml) plus LPS (100 ng/ml). (d) Effect of TFRG on expression of miRNA-155 in stimulated RAW264.7 cells. The cells were stimulated with LPS plus IFN- γ only or stimulated with different concentrations (20, 40, 60 μ g/ml) of TFRG for 18 h. Total RNA was isolated and the expression of miRNA-155 was determined by qPCR. RNU6B was used here as an endogenous control. The data represent the mean ± SEM of triplicate experiments. ∗ P
    Nf κ B P50 P65 Ez Tfa Transcription Factor Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    3-Cl-AHPC-mediated phosphorylation of IKK α and IKK β , and activation of the NF- κ B canonical and noncanonical pathways. ( a ) 3-Cl-AHPC activates NF- κ B in KG-1 cells; cells were transduced with lentiviral NF- κ B reporter (GFP) particles transiently for 48 h and then treated with 1 μ M 3-Cl-AHPC for 24 h. ( b ) NF- κ B activation inhibitor JSH-23 blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μ M 3-Cl-AHPC for 24 h. ( c ) 3-Cl-AHPC enhances phosphorylation of IKK α and IKK β . ( d ) 3-Cl-AHPC induces phosphorylation of NF- κ Bp65/RelA at Ser276. ( e ) 3-Cl-AHPC enhances phosphorylation of NF- κ B2p100 followed by processing to p52 in both cell lines. ( f ) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF- κ B2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μ M) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. * and ** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively ( P -value is

    Journal: Cell Death and Differentiation

    Article Title: Maximal adamantyl-substituted retinoid-related molecule-induced apoptosis requires NF-κB noncanonical and canonical pathway activation

    doi: 10.1038/cdd.2010.84

    Figure Lengend Snippet: 3-Cl-AHPC-mediated phosphorylation of IKK α and IKK β , and activation of the NF- κ B canonical and noncanonical pathways. ( a ) 3-Cl-AHPC activates NF- κ B in KG-1 cells; cells were transduced with lentiviral NF- κ B reporter (GFP) particles transiently for 48 h and then treated with 1 μ M 3-Cl-AHPC for 24 h. ( b ) NF- κ B activation inhibitor JSH-23 blocks 3-Cl-AHPC-mediated apoptosis. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI-positive cells) and lower right (early apoptotic cells, annexin V positive, PI-negative) quadrants. KG-1 cells were exposed to 1 μ M 3-Cl-AHPC for 24 h. ( c ) 3-Cl-AHPC enhances phosphorylation of IKK α and IKK β . ( d ) 3-Cl-AHPC induces phosphorylation of NF- κ Bp65/RelA at Ser276. ( e ) 3-Cl-AHPC enhances phosphorylation of NF- κ B2p100 followed by processing to p52 in both cell lines. ( f ) 3-Cl-AHPC induces increased RelB expression and the RelB binding with NF- κ B2p100/p52. Cells were grown and exposed to vehicle or 3-Cl-AHPC (1.0 μ M) for various times as described in Materials and Methods section. Columns represent mean of two independent experiments. Error bars indicate standard deviations. * and ** significantly different from control cells; and JSH-23+3-Cl-AHPC from 3-Cl-AHPC treated cells, respectively ( P -value is

    Article Snippet: NF- κ B activation inhibitor II JSH-23, proteasome inhibitor MG132 and lysosomal inhibitor CA-074Me from EMD Biosciences (Gibbstown, NJ, USA); and Cdc37 shRNA expression vector from Open Biosystems (Frederick, MD, USA).

    Techniques: Activation Assay, Transduction, Labeling, Staining, Expressing, Binding Assay

    Reducing basal NF- κ B activity upregulates Hgf expression in MDSCs. ( a ) Real-time RT-PCR analysis revealed that Hgf was significantly upregulated in p65 +/− compared with WT MDSCs (* P

    Journal: Cell Death & Disease

    Article Title: NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair

    doi: 10.1038/cddis.2015.66

    Figure Lengend Snippet: Reducing basal NF- κ B activity upregulates Hgf expression in MDSCs. ( a ) Real-time RT-PCR analysis revealed that Hgf was significantly upregulated in p65 +/− compared with WT MDSCs (* P

    Article Snippet: To block NF-κ B activation, WT MDSCs were treated with the reversible ATP-competitive inhibitor of IKKβ , IKKi (EMD Millipore, Billerca, MA, USA) at 5 μ M. Recombinant mouse HGF was used at a concentration of 100 ng/ml (Pepro Tech, Rocky Hill, NJ, USA).

    Techniques: Activity Assay, Expressing, Quantitative RT-PCR

    Effect of TFRG on NF-κB, MAPK activities, and microRNA-155 expression in LPS/IFNγ-stimulated RAW264.7 cells . (a, b) DNA binding activity of p50 and p65 proteins in nuclear extracts was assessed using NF- κ Bp50/p65 EZ-TFA transcription factor assay. Absorbance was measured at 450 nm in a microplate spectrophotometer. Results were normalized to absorbance/mg protein. (c) RAW264.7 cells were plated at a density of 1 × 10 6 cells/well in 30-mm dish overnight. TFRG were added to cells for 1 h followed by 30-min stimulation of IFN γ (10 U/ml) plus LPS (100 ng/ml). (d) Effect of TFRG on expression of miRNA-155 in stimulated RAW264.7 cells. The cells were stimulated with LPS plus IFN- γ only or stimulated with different concentrations (20, 40, 60 μ g/ml) of TFRG for 18 h. Total RNA was isolated and the expression of miRNA-155 was determined by qPCR. RNU6B was used here as an endogenous control. The data represent the mean ± SEM of triplicate experiments. ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Total Flavonoids from Radix Glycyrrhiza Exert Anti-Inflammatory and Antitumorigenic Effects by Inactivating iNOS Signaling Pathways

    doi: 10.1155/2018/6714282

    Figure Lengend Snippet: Effect of TFRG on NF-κB, MAPK activities, and microRNA-155 expression in LPS/IFNγ-stimulated RAW264.7 cells . (a, b) DNA binding activity of p50 and p65 proteins in nuclear extracts was assessed using NF- κ Bp50/p65 EZ-TFA transcription factor assay. Absorbance was measured at 450 nm in a microplate spectrophotometer. Results were normalized to absorbance/mg protein. (c) RAW264.7 cells were plated at a density of 1 × 10 6 cells/well in 30-mm dish overnight. TFRG were added to cells for 1 h followed by 30-min stimulation of IFN γ (10 U/ml) plus LPS (100 ng/ml). (d) Effect of TFRG on expression of miRNA-155 in stimulated RAW264.7 cells. The cells were stimulated with LPS plus IFN- γ only or stimulated with different concentrations (20, 40, 60 μ g/ml) of TFRG for 18 h. Total RNA was isolated and the expression of miRNA-155 was determined by qPCR. RNU6B was used here as an endogenous control. The data represent the mean ± SEM of triplicate experiments. ∗ P

    Article Snippet: ELISA for NF-κ B p50/p65 EZ-TFA transcription factor assay kit was purchased from Millipore (Bedford, MA, USA).

    Techniques: Expressing, Binding Assay, Activity Assay, Transcription Factor Assay, Spectrophotometry, Isolation, Real-time Polymerase Chain Reaction