Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Vrt||ICC, IF, WB, ChIP, IHC||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain for 1 year at 2 to 8°C from date of shipment. For maximum recovery of product, centrifuge the original vial prior to removing the cap.|
|Material Size||200 µg|
|Reference overview||Application||Species||Pub Med ID|
|In vivo measurement of dose distribution in patients' lymphocytes: helical tomotherapy versus step-and-shoot IMRT in prostate cancer.|
Zwicker, F; Swartman, B; Roeder, F; Sterzing, F; Hauswald, H; Thieke, C; Weber, KJ; Huber, PE; Schubert, K; Debus, J; Herfarth, K
Journal of radiation research 56 239-47 2015
In radiotherapy, in vivo measurement of dose distribution within patients' lymphocytes can be performed by detecting gamma-H2AX foci in lymphocyte nuclei. This method can help in determining the whole-body dose. Options for risk estimations for toxicities in normal tissue and for the incidence of secondary malignancy are still under debate. In this investigation, helical tomotherapy (TOMO) is compared with step-and-shoot IMRT (SSIMRT) of the prostate gland by measuring the dose distribution within patients' lymphocytes. In this prospective study, blood was taken from 20 patients before and 10 min after their first irradiation fraction for each technique. The isolated leukocytes were fixed 2 h after radiation. DNA double-stranded breaks in lymphocyte nuclei were stained immunocytochemically using anti-gamma-H2AX antibodies. Gamma-H2AX foci distribution in lymphocytes was determined for each patient. Using a calibration line, dose distributions in patients' lymphocytes were determined by studying the gamma-H2AX foci distribution, and these data were used to generate a cumulative dose-lymphocyte histogram (DLH). Measured in vivo (DLH), significantly fewer lymphocytes indicated low-dose exposure (less than 40% of the applied dose) during TOMO compared with SSIMRT. The dose exposure range, between 45 and 100%, was equal with both radiation techniques. The mean number of gamma-H2AX foci per lymphocyte was significantly lower in the TOMO group compared with the SSIMRT group. In radiotherapy of the prostate gland, TOMO generates a smaller fraction of patients' lymphocytes with low-dose exposure relative to the whole body compared with SSIMRT. Differences in the constructional buildup of the different linear accelerator systems, e.g. the flattening filter, may be the cause thereof. The influence of these methods on the incidence of secondary malignancy should be investigated in further studies.
|DNA damage induces nuclear actin filament assembly by Formin -2 and Spire-½ that promotes efficient DNA repair. [corrected].|
Belin, BJ; Lee, T; Mullins, RD
eLife 4 e07735 2015
Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments-detectable by phalloidin and live-cell actin probes-with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.
|Limiting replication stress during somatic cell reprogramming reduces genomic instability in induced pluripotent stem cells.|
Ruiz, S; Lopez-Contreras, AJ; Gabut, M; Marion, RM; Gutierrez-Martinez, P; Bua, S; Ramirez, O; Olalde, I; Rodrigo-Perez, S; Li, H; Marques-Bonet, T; Serrano, M; Blasco, MA; Batada, NN; Fernandez-Capetillo, O
Nature communications 6 8036 2015
The generation of induced pluripotent stem cells (iPSC) from adult somatic cells is one of the most remarkable discoveries in recent decades. However, several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Here we show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. Our data reveal that lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC.
|Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells.|
Barazzuol, L; Rickett, N; Ju, L; Jeggo, PA
Journal of cell science 128 3597-606 2015
The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C)), ataxia telangiectasia mutated (Atm(-/-)) and double mutant Atm(-/-)/Lig4(Y288C) mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4(Y288C) mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4(Y288C) mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4(Y288C) mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.
|Progerin reduces LAP2α-telomere association in Hutchinson-Gilford progeria.|
Chojnowski, A; Ong, PF; Wong, ES; Lim, JS; Mutalif, RA; Navasankari, R; Dutta, B; Yang, H; Liow, YY; Sze, SK; Boudier, T; Wright, GD; Colman, A; Burke, B; Stewart, CL; Dreesen, O
eLife 4 2015
Hutchinson-Gilford progeria (HGPS) is a premature ageing syndrome caused by a mutation in LMNA, resulting in a truncated form of lamin A called progerin. Progerin triggers loss of the heterochromatic marker H3K27me3, and premature senescence, which is prevented by telomerase. However, the mechanism how progerin causes disease remains unclear. Here, we describe an inducible cellular system to model HGPS and find that LAP2α (lamina-associated polypeptide-α) interacts with lamin A, while its interaction with progerin is significantly reduced. Super-resolution microscopy revealed that over 50% of telomeres localize to the lamina and that LAP2α association with telomeres is impaired in HGPS. This impaired interaction is central to HGPS since increasing LAP2α levels rescues progerin-induced proliferation defects and loss of H3K27me3, whereas lowering LAP2 levels exacerbates progerin-induced defects. These findings provide novel insights into the pathophysiology underlying HGPS, and how the nuclear lamina regulates proliferation and chromatin organization.
|Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.|
Baker, CL; Petkova, P; Walker, M; Flachs, P; Mihola, O; Trachtulec, Z; Petkov, PM; Paigen, K
PLoS genetics 11 e1005512 2015
Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.
|Syringaresinol protects against hypoxia/reoxygenation-induced cardiomyocytes injury and death by destabilization of HIF-1α in a FOXO3-dependent mechanism.|
Cho, S; Cho, M; Kim, J; Kaeberlein, M; Lee, SJ; Suh, Y
Oncotarget 6 43-55 2015
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of hypoxic response and has been a prime therapeutic target for ischemia/reperfusion (I/R)-derived myocardial dysfunction and tissue damage. There is also increasing evidence that HIF-1 plays a central role in regulating aging, both through interactions with key longevity factors including Sirtuins and mTOR, as well as by directly promoting longevity in Caenorhabditis elegans.We investigated a novel function and the underlying mechanism of syringaresinol, a lignan compound, in modulation of HIF-1 and protection against cellular damage and death in a cardiomyocyte model of I/R injury. Syringaresinol caused destabilization of HIF-1α following H/R and then protected against hypoxia/reoxygenation (H/R)-induced cellular damage, apoptosis, and mitochondrial dysfunction in a dose-dependent manner. Knock-down of FOXO3 by specific siRNAs completely abolished the ability of syringaresinol to inhibit HIF-1 stabilization and apoptosis caused by H/R. Syringaresinol stimulated the nuclear localization and activity of FOXO3 leading to increased expression of antioxidant genes and decreased levels of reactive oxygen species (ROS) following H/R. Our results provide a new mechanistic insight into a functional role of syringaresinol against H/R-induced cardiomyocyte injury and death. The degradation of HIF-1α through activation of FOXO3 is a potential therapeutic strategy for ischemia-related diseases.
|Fluorescence Activated Cell Sorting (FACS)||25415049|
|The SMAC mimetic BV6 sensitizes colorectal cancer cells to ionizing radiation by interfering with DNA repair processes and enhancing apoptosis.|
Hehlgans, S; Oppermann, J; Reichert, S; Fulda, S; Rödel, C; Rödel, F
Radiation oncology (London, England) 10 198 2015
In the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells.Colorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0-4 μM) with or without irradiation (2-8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)).BV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells.The SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells.
|Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.|
Lin, JR; Fallahi-Sichani, M; Sorger, PK
Nature communications 6 8390 2015
Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.
|The SET Domain Is Essential for Metnase Functions in Replication Restart and the 5' End of SS-Overhang Cleavage.|
Kim, HS; Kim, SK; Hromas, R; Lee, SH
PloS one 10 e0139418 2015
Metnase (also known as SETMAR) is a chimeric SET-transposase protein that plays essential role(s) in non-homologous end joining (NHEJ) repair and replication fork restart. Although the SET domain possesses histone H3 lysine 36 dimethylation (H3K36me2) activity associated with an improved association of early repair components for NHEJ, its role in replication restart is less clear. Here we show that the SET domain is necessary for the recovery from DNA damage at the replication forks following hydroxyurea (HU) treatment. Cells overexpressing the SET deletion mutant caused a delay in fork restart after HU release. Our In vitro study revealed that the SET domain but not the H3K36me2 activity is required for the 5' end of ss-overhang cleavage with fork and non-fork DNA without affecting the Metnase-DNA interaction. Together, our results suggest that the Metnase SET domain has a positive role in restart of replication fork and the 5' end of ss-overhang cleavage, providing a new insight into the functional interaction of the SET and the transposase domains.
|Advancing cancer research: From hallmarks & biomarkers to tumor microenvironment progression|
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics (EMD)|
|How can I check the phosphospecificity of my antibody once my protein is already immobilized on a membrane?||You can check for the phosphospecificity of your antibody using Lambda Protein Phosphatase. Simply perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate and transfer the proteins to your membrane of choice. Wash the blotted nitrocellulose twice with water. Block the blotted nitrocellulose in freshly prepared TBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 hour at 20-25°C with constant agitation. Incubate the nitrocellulose in TBS containing 1% bovine serum albumin (BSA), 0.1% Triton X-100 and 2 mM MnCl2, and where dephosphorylation of proteins is desirable, 400 U/ml Lambda Protein Phosphatase for two hours at room temperature, or overnight at 4°C. After incubation, wash the nitrocellulose in PBS-0.1% Tween 20 for 3-5 minutes. Rinse the nitrocellulose in 4-5 changes of water. Continue with your western blotting assay.|