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  • Nuclear factor-κB (NF-κB) inhibitory protein IκBβ determines apoptotic cell death following exposure to oxidative stress. 22223647

    The transcription factor NF-κB regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-κB activity following exposure to oxidative stress, the role of the IκB family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of IκBβ in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-κB signaling through IκBβ would prevent apoptotic cell death. Using IκBβ knock-in mice (AKBI), in which the IκBα gene is replaced with the IκBβ cDNA, we show that IκBβ overexpression prevented oxidative stress-induced apoptotic cell death. This was associated with retention of NF-κB subunits in the nucleus and maintenance of NF-κB activity. Furthermore, the up-regulation of pro-apoptotic genes in WT murine embryonic fibroblasts (MEFs) exposed to serum starvation was abrogated in AKBI MEFs. Inhibition of apoptosis was observed in WT MEFs overexpressing IκBβ with simultaneous IκBα knockdown, whereas IκBβ overexpression alone did not produce this effect. These findings represent a necessary but not sufficient role of IκBβ in preventing oxidant stress-induced cell death.
    Document Type:
    Reference
    Product Catalog Number:
    05-829
    Product Catalog Name:
    Anti-α-Tubulin Antibody, clone DM1A

  • Chronic epithelial NF-κB activation accelerates APC loss and intestinal tumor initiation through iNOS up-regulation. 22893683

    The role of NF-κB activation in tumor initiation has not been thoroughly investigated. We generated Ikkβ(EE)(IEC) transgenic mice expressing constitutively active IκB kinase β (IKKβ) in intestinal epithelial cells (IECs). Despite absence of destructive colonic inflammation, Ikkβ(EE)(IEC) mice developed intestinal tumors after a long latency. However, when crossed to mice with IEC-specific allelic deletion of the adenomatous polyposis coli (Apc) tumor suppressor locus, Ikkβ(EE)(IEC) mice exhibited more β-catenin(+) early lesions and visible small intestinal and colonic tumors relative to Apc(+/ΔIEC) mice, and their survival was severely compromised. IEC of Ikkβ(EE)(IEC) mice expressed high amounts of inducible nitric oxide synthase (iNOS) and elevated DNA damage markers and contained more oxidative DNA lesions. Treatment of Ikkβ(EE)(IEC)/Apc(+/ΔIEC) mice with an iNOS inhibitor decreased DNA damage markers and reduced early β-catenin(+) lesions and tumor load. The results suggest that persistent NF-κB activation in IEC may accelerate loss of heterozygocity by enhancing nitrosative DNA damage.
    Document Type:
    Reference
    Product Catalog Number:
    06-599
    Product Catalog Name:
    Anti-acetyl-Histone H3 Antibody

  • Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10. 23575687

    Adenosine diphosphate-ribosylation is a post-translational modification mediated by intracellular and membrane-associated extracellular enzymes and many bacterial toxins. The intracellular enzymes modify their substrates either by poly-ADP-ribosylation, exemplified by ARTD1/PARP1, or by mono-ADP-ribosylation. The latter has been discovered only recently, and little is known about its physiological relevance. The founding member of mono-ADP-ribosyltransferases is ARTD10/PARP10. It possesses two ubiquitin-interaction motifs, a unique feature among ARTD/PARP enzymes. Here, we find that the ARTD10 ubiquitin-interaction motifs bind to K63-linked poly-ubiquitin, a modification that is essential for NF-κB signalling. We therefore studied the role of ARTD10 in this pathway. ARTD10 inhibits the activation of NF-κB and downstream target genes in response to interleukin-1β and tumour necrosis factor-α, dependent on catalytic activity and poly-ubiquitin binding of ARTD10. Mechanistically ARTD10 interferes with poly-ubiquitination of NEMO, which interacts with and is a substrate of ARTD10. Our findings identify a novel regulator of NF-κB signalling and provide evidence for cross-talk between K63-linked poly-ubiquitination and mono-ADP-ribosylation.
    Document Type:
    Reference
    Product Catalog Number:
    MABE1044

  • IκBβ-mediated NF-κB activation confers protection against hyperoxic lung injury. 24066808

    Supplemental oxygen is frequently used in an attempt to improve oxygen delivery; however, prolonged exposure results in damage to the pulmonary endothelium and epithelium. Although NF-κB has been identified as a redox-responsive transcription factor, whether NF-κB activation exacerbates or attenuates hyperoxic lung injury is unclear. We determined that sustained NF-κB activity mediated by IκBβ attenuates lung injury and prevents mortality in adult mice exposed to greater than 95% O2. Adult wild-type mice demonstrated evidence of alveolar protein leak and 100% mortality by 6 days of hyperoxic exposure, and showed NF-κB nuclear translocation that terminated after 48 hours. Furthermore, these mice showed increased expression of NF-κB-regulated proinflammatory and proapoptotic cytokines. In contrast, mice overexpressing the NF-κB inhibitory protein, IκBβ (AKBI), demonstrated significant resistance to hyperoxic lung injury, with 50% surviving through 8 days of exposure. This was associated with NF-κB nuclear translocation that persisted through 96 hours of exposure. Although induction of NF-κB-regulated proinflammatory cytokines was not different between wild-type and AKBI mice, significant up-regulation of antiapoptotic proteins (BCL-2, BCL-XL) was found exclusively in AKBI mice. We conclude that sustained NF-κB activity mediated by IκBβ protects against hyperoxic lung injury through increased expression of antiapoptotic genes.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy. 21903858

    BRAF(V600E) is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAF(V600E) mutation, TIMP-1 expression, and NF-κB activation. We found that BRAF(V600E) mutation occurs selectively in PTC nodules and is associated with hyperactivation of NF-κB and upregulation of both TIMP-1 and its receptor CD63. To assess the functional relationship among these factors, we first silenced BRAF gene in BCPAP cells, harboring BRAF(V600E) mutation. We found that silencing causes a marked decrease in TIMP-1 expression and NF-κB binding activity, as well as decreased invasiveness. After treatment with specific inhibitors of MAPK pathway, we found that only sorafenib was able to increase IκB-α and reduce both TIMP-1 expression and Akt phosphorylation in BCPAP cells, indicating that BRAF(V600E) activates NF-κB and this pathway is MEK-independent. Taken together, our findings demonstrate that BRAF(V600E) causes upregulation of TIMP-1 via NF-κB. TIMP-1 binds then its surface receptor CD63, leading eventually to Akt activation, which in turn confers antiapoptotic behavior and promotion of cell invasion. The recognition of this functional trilogy provides insight on how BRAF(V600E) determines cancer initiation, progression, and invasiveness in PTC, also identifying new therapeutic targets for the treatment of highly aggressive forms.
    Document Type:
    Reference
    Product Catalog Number:
    07-146
    Product Catalog Name:
    Anti-Histone H2A (acidic patch) Antibody

  • Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies. 21999414

    The characterization and cellular localization of transcription factors like NF-κB requires the use of antibodies for western blots and immunohistochemistry. However, if target protein levels are low and the antibodies not well characterized, false positive data can result. In studies of NF-κB activity in the CNS, antibodies detecting NF-κB proteins have been used to support the finding that NF-κB is constitutively active in neurons, and activity levels are further increased by neurotoxic treatments, glutamate stimulation, or elevated synaptic activity. The specificity of the antibodies used was analyzed in this study.Selectivity and nonselectivity of commonly used commercial and non-commercial p50 and p65 antibodies were demonstrated in western blot assays conducted in tissues from mutant gene knockout mice lacking the target proteins.A few antibodies for p50 and p65 each mark a single band at the appropriate molecular weight in gels containing proteins from wildtype tissue, and this band is absent in proteins from knockout tissues. Several antibodies mark proteins that are present in knockout tissues, indicating that they are nonspecific. These include antibodies raised against the peptide sequence containing the nuclear localization signals of p65 (MAB3026; Chemicon) and p50 (sc-114; Santa Cruz). Some antibodies that recognize target proteins at the correct molecular weight still fail in western blot analysis because they also mark additional proteins and inconsistently so. We show that the criterion for validation by use of blocking peptides can still fail the test of specificity, as demonstrated for several antibodies raised against p65 phosphorylated at serine 276. Finally, even antibodies that show specificity in western blots produce nonspecific neuronal staining by immunohistochemistry.We note that many of the findings in the literature about neuronal NF-κB are based on data garnered with antibodies that are not selective for the NF-κB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-κB activity in the brain.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The PRKD1 promoter is a target of the KRas-NF-κB pathway in pancreatic cancer. 27649783

    Increased expression of PRKD1 and its gene product protein kinase D1 (PKD1) are linked to oncogenic signaling in pancreatic ductal adenocarcinoma, but a direct functional relationship to oncogenic KRas has not been established so far. We here describe the PRKD1 gene promoter as a target for oncogenic KRas signaling. We demonstrate that KRas-induced activation of the canonical NF-κB pathway is one mechanism of how PRKD1 expression is increased and identify the binding sites for NF-κB in the PRKD1 promoter. Altogether, these results describe a novel mechanism governing PRKD1 gene expression in PDA and provide a functional link between oncogenic KRas, NF-κB and expression of PRKD1.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Par-complex aPKC and Par3 cross-talk with innate immunity NF-κB pathway in epithelial cells. 24244864

    Components of the Par-complex, atypical PKC and Par3, have been found to be downregulated upon activation of NF-κB in intestinal epithelial cells. To determine their possible role in pro-inflammatory responses we transduced Caco-2 human colon carcinoma cells with constitutively active (ca) PKCι or anti-Par3 shRNA-expressing lentiviral particles. Contrary to previous reports in other cell types, ca-PKCι did not activate, but rather decreased, baseline NF-κB activity in a luminiscence reporter assay. An identical observation applied to a PB1 domain deletion PKCι, which fails to localize to the tight-junction. Conversely, as expected, the same ca-PKCι activated NF-κB in non-polarized HEK293 cells. Likewise, knockdown of Par3 increased NF-κB activity and, surprisingly, greatly enhanced its response to TNFα, as shown by transcription of IL-8, GRO-1, GRO-2 and GRO-3. We conclude that aPKC and Par3 are inhibitors of the canonical NF-κB activation pathway, although perhaps acting through independent pathways, and may be involved in pro-inflammatory responses.
    Document Type:
    Reference
    Product Catalog Number:
    07-330
    Product Catalog Name:
    Anti-Partitioning-defective 3 Antibody

  • GMI, a Ganoderma immunomodulatory protein, down-regulates tumor necrosis factor α-induced expression of matrix metalloproteinase 9 via NF-κB pathway in human alveolar epi ... 21028821

    Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-κB. 23087373

    Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use.
    Document Type:
    Reference
    Product Catalog Number:
    06-599
    Product Catalog Name:
    Anti-acetyl-Histone H3 Antibody