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|Description||Magna ChIP™ A/G Chromatin Immunoprecipitation Kit|
|Overview||Unlike standard ChIP protocols that can be laborious and time consuming, the Magna ChIP kit protocol can reduce the amount of time required to perform a ChIP experiment from three days to one. Additionally, the smaller Magna ChIP reaction volume increases the relative concentration of the antibody enabling the ChIP reaction to be performed with reduced amounts of both antibody and sheared chromatin. Finally because this kit uses a blend of protein A and protein G beads, a wider range of antibody isotypes can be used than A or G alone. This allows a wider variety of antibodies to be used and avoids the need to purchase separate kits for protein A and protein G based immunoprecipitation. Because Magna ChIP kits use paramagnetic beads they are compatible with automated high throughput platforms, thus allowing a large number of ChIP reactions to be carried out simultaneously. Features & Benefits:
Chromatin Immunoprecipitation (ChIP) is an important technique allowing the analysis of in vivo interactions of proteins with genomic DNA. Any chromatin-associated or DNA binding protein can be analyzed with this technique, provided a good antibody to the protein exists. One can measure different proteins localized to a specific region of the genome, or the genome wide distribution of a specific protein. Another powerful application of this technique is to analyze changes in histone modifications that correlate with processes like transcription, mitosis or DNA repair.
|Background Information||Chromatin Immunoprecipitation (ChIP) is a powerful technique for mapping the in vivo distribution of proteins associated with chromosomal DNA. These proteins can be histone subunits and post-translational modifications or other chromatin associated proteins such as transcription factors, chromatin regulators, etc. Additionally, ChIP can be used to identify regions of the genome associated with these proteins, or conversely, to identify proteins associated with a particular region of the genome. ChIP methodology often involves protein-DNA and protein-protein cross-linking, fragmentation of the cross-linked chromatin, and subsequent immunoprecipitation of chromatin with an antibody specific to a target protein. The DNA fragments isolated in complex with the target protein can be identified by a variety of methods including PCR, DNA microarray and DNA sequencing. Standard or quantitative PCR can be performed to verify whether a particular DNA sequence (the gene or region of the genome) is associated with the protein of interest. The combination of ChIP and promoter or genomic tiling microarrays (ChIP-chip) allows genome-wide identification of DNA-binding sites for chromatin-associated proteins with precise resolution. Alternatively, high-throughput sequencing of libraries constructed from immunoprecipitated chromosomal DNA (ChIP-Seq) is a powerful alternative to ChIP-chip in mapping the protein-DNA interactions across mammalian genomes.|
|Materials Required but Not Delivered||Magna Grip™ Rack 8 well ( 20-400) (Now Available!) or similar magnetic rack.|
|Application||Single day chromatin immunoprecipitation (ChIP) kit containing all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using magnetic A/G beads.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Upon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 1 year from date of shipment when stored as directed.|
|Material Size||22 assays|
|Material Package||Kit capacity: 22 chromatin immunoprecipitation assays|
|Reference overview||Application||Species||Pub Med ID|
|The role of SMAD3 in the genetic predisposition to papillary thyroid carcinoma.|
Wang, Y; He, H; Liyanarachchi, S; Genutis, LK; Li, W; Yu, L; Phay, JE; Shen, R; Brock, P; de la Chapelle, A
Genet Med 0 2018
PurposeTo identify and characterize the functional variants, regulatory gene networks, and potential binding targets of SMAD3 in the 15q22 thyroid cancer risk locus.MethodsWe performed linkage disequilibrium (LD) and haplotype analyses to fine map the 15q22 locus. Luciferase reporter assays were applied to evaluate the regulatory effects of the candidate variants. Knockdown by small interfering RNA, microarray analysis, chromatin immunoprecipitation (ChIP) and quantitative real-time polymerase chain reaction assays were performed to reveal the regulatory gene network and identify its binding targets.ResultsWe report a 25.6-kb haplotype within SMAD3 containing numerous single-nucleotide polymorphisms (SNPs) in high LD. SNPs rs17293632 and rs4562997 were identified as functional variants of SMAD3 by luciferase assays within the LD region. These variants regulate SMAD3 transcription in an allele-specific manner through enhancer elements in introns of SMAD3. Knockdown of SMAD3 in thyroid cancer cell lines revealed its regulatory gene network including two upregulated genes, SPRY4 and SPRY4-IT1. Sequence analysis and ChIP assays validated the actual binding of SMAD3 protein to multiple SMAD binding element sites in the region upstream of SPRY4.ConclusionOur data provide a functional annotation of the 15q22 thyroid cancer risk locus.GENETICS in MEDICINE advance online publication, 4 January 2018; doi:10.1038/gim.2017.224.
|Pharmacologic Inhibition of the Menin-MLL Interaction Leads to Transcriptional Repression of PEG10 and Blocks Hepatocellular Carcinoma.|
Kempinska, K; Malik, B; Borkin, D; Klossowski, S; Shukla, S; Miao, H; Wang, J; Cierpicki, T; Grembecka, J
Mol Cancer Ther 17 26-38 2018
Hepatocellular carcinoma (HCC) accounts for approximately 85% of malignant liver tumors and results in 600,000 deaths each year, emphasizing the need for new therapies. Upregulation of menin was reported in HCC patients and high levels of menin correlate with poor patient prognosis. The protein-protein interaction between menin and histone methyltransferase mixed lineage leukemia 1 (MLL1) plays an important role in the development of HCC, implying that pharmacologic inhibition of this interaction could lead to new therapeutic strategy for the HCC patients. Here, we demonstrate that the menin-MLL inhibitor MI-503 shows antitumor activity in in vitro and in vivo models of HCC and reveals the potential mechanism of menin contribution to HCC. Treatment with MI-503 selectively kills various HCC cell lines and this effect is significantly enhanced by a combination of MI-503 with sorafenib, the standard-of-care therapy for HCC. Furthermore, MI-503 reduces sphere formation and cell migration in in vitro HCC models. When applied in vivo, MI-503 gives a strong antitumor effect both as a single agent and in combination with sorafenib in mice xenograft models of HCC. Mechanistically, treatment with MI-503 downregulates expression of several genes known to play a critical role in proliferation and migration of HCC cells, including PEG10, and displaces the menin-MLL1 complex from the PEG10 promoter, resulting in reduced H3K4 methylation and transcriptional repression. Overall, our studies reveal a mechanistic link between menin and genes involved in HCC and demonstrate that pharmacologic inhibition of the menin-MLL interaction might represent a promising therapeutic approach for HCC. Mol Cancer Ther; 17(1); 26-38. ©2017 AACR.
|Actomyosin-Mediated Tension Orchestrates Uncoupled Respiration in Adipose Tissues.|
Tharp, KM; Kang, MS; Timblin, GA; Dempersmier, J; Dempsey, GE; Zushin, PH; Benavides, J; Choi, C; Li, CX; Jha, AK; Kajimura, S; Healy, KE; Sul, HS; Saijo, K; Kumar, S; Stahl, A
Cell Metab 27 602-615.e4 2018
The activation of brown/beige adipose tissue (BAT) metabolism and the induction of uncoupling protein 1 (UCP1) expression are essential for BAT-based strategies to improve metabolic homeostasis. Here, we demonstrate that BAT utilizes actomyosin machinery to generate tensional responses following adrenergic stimulation, similar to muscle tissues. The activation of actomyosin mechanics is critical for the acute induction of oxidative metabolism and uncoupled respiration in UCP1+ adipocytes. Moreover, we show that actomyosin-mediated elasticity regulates the thermogenic capacity of adipocytes via the mechanosensitive transcriptional co-activators YAP and TAZ, which are indispensable for normal BAT function. These biomechanical signaling mechanisms may inform future strategies to promote the expansion and activation of brown/beige adipocytes.
|Zeb1-Hdac2-eNOS circuitry identifies early cardiovascular precursors in naive mouse embryonic stem cells.|
Cencioni, C; Spallotta, F; Savoia, M; Kuenne, C; Guenther, S; Re, A; Wingert, S; Rehage, M; Sürün, D; Siragusa, M; Smith, JG; Schnütgen, F; von Melchner, H; Rieger, MA; Martelli, F; Riccio, A; Fleming, I; Braun, T; Zeiher, AM; Farsetti, A; Gaetano, C
Nat Commun 9 1281 2018
Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells (mESC) and occurs after release from serum and leukemia inhibitory factor (LIF). Here we show that after release from pluripotency, a subpopulation of mESC, kept in the naive state by 2i/LIF, expresses endothelial nitric oxide synthase (eNOS) and endogenously synthesizes NO. This eNOS/NO-positive subpopulation (ESNO+) expresses mesendodermal markers and is more efficient in the generation of cardiovascular precursors than eNOS/NO-negative cells. Mechanistically, production of endogenous NO triggers rapid Hdac2 S-nitrosylation, which reduces association of Hdac2 with the transcriptional repression factor Zeb1, allowing mesendodermal gene expression. In conclusion, our results suggest that the interaction between Zeb1, Hdac2, and eNOS is required for early mesendodermal differentiation of naive mESC.
|Cold atmospheric plasma as a potential tool for multiple myeloma treatment.|
Xu, D; Xu, Y; Cui, Q; Liu, D; Liu, Z; Wang, X; Yang, Y; Feng, M; Liang, R; Chen, H; Ye, K; Kong, MG
Oncotarget 9 18002-18017 2018
Multiple myeloma (MM) is a fatal and incurable hematological malignancy thus new therapy need to be developed. Cold atmospheric plasma, a new technology that could generate various active species, could efficiently induce various tumor cells apoptosis. More details about the interaction of plasma and tumor cells need to be addressed before the application of gas plasma in clinical cancer treatment. In this study, we demonstrate that He+O2 plasma could efficiently induce myeloma cell apoptosis through the activation of CD95 and downstream caspase cascades. Extracellular and intracellular reactive oxygen species (ROS) accumulation is essential for CD95-mediated cell apoptosis in response to plasma treatment. Furthermore, p53 is shown to be a key transcription factor in activating CD95 and caspase cascades. More importantly, we demonstrate that CD95 expression is higher in tumor cells than in normal cells in both MM cell lines and MM clinical samples, which suggests that CD95 could be a favorable target for plasma treatment as it could selectively inactivate myeloma tumor cells. Our results illustrate the molecular details of plasma induced myeloma cell apoptosis and it shows that gas plasma could be a potential tool for myeloma therapy in the future.
|C/EBPδ drives interactions between human MAIT cells and endothelial cells that are important for extravasation.|
Lee, CH; Zhang, HH; Singh, SP; Koo, L; Kabat, J; Tsang, H; Singh, TP; Farber, JM
Elife 7 2018
Many mediators and regulators of extravasation by bona fide human memory-phenotype T cells remain undefined. Mucosal-associated invariant T (MAIT) cells are innate-like, antibacterial cells that we found excelled at crossing inflamed endothelium. They displayed abundant selectin ligands, with high expression of FUT7 and ST3GAL4, and expressed CCR6, CCR5, and CCR2, which played non-redundant roles in trafficking on activated endothelial cells. MAIT cells selectively expressed CCAAT/enhancer-binding protein delta (C/EBPδ). Knockdown of C/EBPδ diminished expression of FUT7, ST3GAL4 and CCR6, decreasing MAIT cell rolling and arrest, and consequently the cells' ability to cross an endothelial monolayer in vitro and extravasate in mice. Nonetheless, knockdown of C/EBPδ did not affect CCR2, which was important for the step of transendothelial migration. Thus, MAIT cells demonstrate a program for extravasastion that includes, in part, C/EBPδ and C/EBPδ-regulated genes, and that could be used to enhance, or targeted to inhibit T cell recruitment into inflamed tissue.
|TCF7L2 positively regulates aerobic glycolysis via the EGLN2/HIF-1α axis and indicates prognosis in pancreatic cancer.|
Xiang, J; Hu, Q; Qin, Y; Ji, S; Xu, W; Liu, W; Shi, S; Liang, C; Liu, J; Meng, Q; Liang, D; Ni, Q; Xu, J; Zhang, B; Yu, X
Cell Death Dis 9 321 2018
Patients with pancreatic ductal adenocarcinoma have much worse prognoses, and much effort has been directed toward understanding the molecular biological aspects of this disease. Accumulated evidence suggests that constitutive activation of the Wnt/β-catenin signalling contributes to the oncogenesis and progression of pancreatic cancer. Transcription factor 7-like2/transcription factor 4 (TCF7L2/TCF4), a β-catenin transcriptional partner, plays a vital role in the Wnt/β-catenin signalling pathway. In the present study, we investigated the clinicopathological significance of TCF7L2 in pancreatic cancer. Our results demonstrated that patients with higher TCF7L2 expression had worse prognosis. Our in vitro studies demonstrated that TCF7L2 positively regulated aerobic glycolysis by suppressing Egl-9 family hypoxia inducible factor 2 (EGLN2), leading to upregulation of hypoxia inducible factor 1 alpha subunit (HIF-1α). The impact of TCF7L2 on aerobic glycolysis was further confirmed in vivo by assessing 18FDG uptake in pancreatic cancer patients and in a subcutaneous xenograft mouse model. In summary, we identified novel predictive markers for prognosis and suggest a previously unrecognized role for TCF7L2 in control of aerobic glycolysis in pancreatic cancer.
|Developmental Decline in the MicroRNA 199a (miR-199a)/miR-214 Cluster in Human Fetal Lung Promotes Type II Cell Differentiation by Upregulating Key Transcription Factors.|
Mishra, R; Benlhabib, H; Guo, W; Lerma Cervantes, CB; Mendelson, CR
Mol Cell Biol 38 2018
The major surfactant protein, SP-A (a product of the SFTPA gene), serves as a marker of type II pneumocyte differentiation and surfactant synthesis. SFTPA expression in cultured human fetal lung (HFL) epithelial cells is upregulated by hormones that increase cyclic AMP (cAMP) and activate TTF-1/NKX2.1 and NF-κB. To further define mechanisms for type II cell differentiation and induction of SP-A, we investigated roles of microRNAs (miRNAs). Using microarray to identify differentially expressed miRNAs in HFL epithelial cells during type II cell differentiation in culture, we observed that members of the miRNA 199a (miR-199a)/miR-214 cluster were significantly downregulated during differentiation. Validated and predicted targets of miR-199a-3p/miR-199a-5p and miR-214, which serve roles in type II cell differentiation (COX-2, NF-κB p50/p65, and CREB1), and the CREB1 target, C/EBPβ, were coordinately upregulated. Accordingly, overexpression of miR-199a-5p, miR-199a-3p, or miR-214 mimics in cultured HFL epithelial cells decreased COX-2, NF-κB p50/p65, CREB1, and C/EBPβ proteins, with an associated inhibition of SP-A expression. Interestingly, overexpression of the EMT factor, ZEB1, which declines during cAMP-induced type II cell differentiation, increased pri-miR-199a and reduced the expression of the targets NF-κB/p50 and COX-2. Collectively, these findings suggest that the developmental decline in miR-199a/miR-214 in HFL causes increased expression of critical targets that enhance type II cell differentiation and SP-A expression.
|Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription.|
Hug, CB; Grimaldi, AG; Kruse, K; Vaquerizas, JM
Cell 169 216-228.e19 2017
Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.
|Disruption of the C/EBPα-miR-182 balance impairs granulocytic differentiation.|
Wurm, AA; Zjablovskaja, P; Kardosova, M; Gerloff, D; Bräuer-Hartmann, D; Katzerke, C; Hartmann, JU; Benoukraf, T; Fricke, S; Hilger, N; Müller, AM; Bill, M; Schwind, S; Tenen, DG; Niederwieser, D; Alberich-Jorda, M; Behre, G
Nat Commun 8 46 2017
Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML.
|An Introduction to Antibodies and Their Applications|
|New Products: Volume 3, 2012|
|Product Selection Guide - Antibodies, small molecule inhibitors, kits, assays and proteins for signaling research.|
|Getting started with ChIP-seq: experimental design to data analysis|
|Magna ChIP protein A/G bead blends simplify the ChIP process and reduce background signals|
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics|
|Magna ChIP™ A/G|