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Search results for: NFATc1 GFP

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#30534207   2018/11/26 To Up

High-performance gene expression and knockout tools using sleeping beauty transposon system.

Similar to retro-/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells. Sleeping Beauty (SB) transposon has adopted for integrating genes into host genomes in recent studies. However, SB-derived vector system for proteins purifying/tracking and gene knockout are still not available.
Kaishun Hu, Yu Li, Wenjing Wu, Hengxing Chen, Zhen Chen, Yin Zhang, Yabin Guo, Yin Dong

1883 related Products with: High-performance gene expression and knockout tools using sleeping beauty transposon system.

112x 100ug11115 μg

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#26959175   2016/03/06 To Up

E-cadherin is important for cell differentiation during osteoclastogenesis.

E-cadherin, a protein responsible for intercellular adhesion between epithelial cells, is also expressed in the monocyte/macrophage lineage. In this study we have explored the involvement of E-cadherin during receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation. Osteoclastogenesis involves a period of precursor expansion followed by multiple fusion events to generate a multinuclear osteoclast that is capable of bone resorption. We asked whether E-cadherin participated in early precursor interactions and recognition or was a component of the osteoclast fusion machinery. Here, we show that endogenous E-cadherin expression is the highest during early stages of osteoclast differentiation, with surface expression visible on small precursor cells (fewer than four nuclei per cell) in both RAW 264.7 cells and primary macrophages. Blocking E-cadherin function with neutralizing antibodies prior to the onset of fusion delayed the expression of TRAP, Cathepsin K, DC-STAMP and NFATc1 and significantly diminished multinucleated osteoclast formation. Conversely, E-cadherin-GFP overexpressing macrophages displayed earlier NFATc1 nuclear translocation along with faster formation of multinucleated osteoclasts compared to control macrophages. Through live imaging we identified that disrupting E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors. The lamellipodium and polarized membrane extensions appeared to be the principal sites of fusion, indicating precursor migration was a critical factor contributing to osteoclast fusion. These findings demonstrate that E-cadherin-mediated cell-cell contacts can modulate osteoclast-specific gene expression and prompt differentiating osteoclast precursors toward migratory and fusion activities.
Cara Fiorino, Rene E Harrison

1006 related Products with: E-cadherin is important for cell differentiation during osteoclastogenesis.

5 x 50 ug50 ug100ug Lyophilized100ug Lyophilized0.1ml (1mg/ml)100ug Lyophilized10 ml30 isolations100ug Lyophilized24 wells100ug Lyophilized

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#26620761   2015/12/01 To Up

Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.
Liren Wang, Yanjiao Shao, Yuting Guan, Liang Li, Lijuan Wu, Fangrui Chen, Meizhen Liu, Huaqing Chen, Yanlin Ma, Xueyun Ma, Mingyao Liu, Dali Li

1400 related Products with: Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

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#24444335   2014/01/21 To Up

Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis.

Matairesinol is a plant lignan present in a wide variety of foodstuffs such as seeds, vegetables and fruits. It has various biological functions including anti-angiogenic, anti-cancer and anti-fungal activities, but its anti-osteoporotic activity, if any, is unknown.
Sik-Won Choi, Kie-In Park, Jeong-Tae Yeon, Byung Jun Ryu, Kwang-Jin Kim, Seong Hwan Kim

2427 related Products with: Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis.

100ug/vial100ug/vial 5 G100 ug/vial100ug/vial100 ug/vial100 ug/vial0.1ml (1mg/ml)100ug/vial100ug/vial100 ug/vial100 ug/vial

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#22585661   // To Up

Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control green fluorescent protein (GFP) gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process.
Toru Ogasawara, Shinsuke Ohba, Fumiko Yano, Hiroshi Kawaguchi, Ung-Il Chung, Tadahito Saito, Yoshiyuki Yonehara, Takashi Nakatsuka, Yoshiyuki Mori, Tsuyoshi Takato, Kazuto Hoshi

2204 related Products with: Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

100ug100ug100ug24 wells100ug100ug100ug Lyophilized200ul100ug Lyophilized100ug100ug100ug

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#21435337   2011/03/22 To Up

Cyclophilin C-associated protein regulation of phagocytic functions via NFAT activation in macrophages.

Experimental cerebral ischemia has been reportedly alleviated by the immunosuppressive agent cyclosporin A (CsA). Cyclophilin C-associated protein (CyCAP) was proposed to be an endogenous equivalent of CsA; CsA- and CyCAP-targeting protein cyclophilin C have attracted extensive attention regarding their ischemia-alleviating mechanisms. In this study we have introduced the specific CyCAP antibody for evaluating its distribution in the rat ischemic brain after middle cerebral artery occlusion. During the recovery of cerebral ischemia in rats, CyCAP was highly expressed in the activated microglia/macrophages in the ischemic lesion. However, it remains unknown what roles CyCAP plays in the activation of macrophage phagocytosis. Thus, we studied CyCAP function using a RAW264.7 macrophage cell line. When we expressed CyCAP-GFP and cyclophilin C-FLAG in RAW264.7 cells, we found that CyCAP and cyclophilin C make a complex, which is competitively inhibited by CsA. Consistently, in immunoprecipitates by anti-calcineurin antibody, cyclophilin C and CyCAP were detected, and CyCAP pulled down NFATc1, suggesting that both CyCAP and cyclophilin C form a complex with calcineurin and NFATc1. When CyCAP was adenovirally overexpressed in RAW cells, NFAT staining increased over the nucleus. Furthermore, calcineurin and IL-2 were increased with time. Thus, CyCAP appears to control macrophage functions by activating NFAT and the resultant IL-2 production. With a protein phosphatase inhibitor PhoSTOP, NFAT was localized more to the cytoplasm, and phagocytosis was decreased strikingly. Thus, we suggest that in a CyCAP-cyclophilin C pathway for macrophage activation, calcineurin phosphatase activity is essential for the phagocytosis activity via dephosphorylation of NFATc1.
Rei Yamaguchi, Masahiro Hosaka, Seiji Torii, Ni Hou, Nobuhito Saito, Yuhei Yoshimoto, Hideaki Imai, Toshiyuki Takeuchi

2610 related Products with: Cyclophilin C-associated protein regulation of phagocytic functions via NFAT activation in macrophages.

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#21153864   2010/12/14 To Up

Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB.

Receptor activator of NF-κB (RANK) is a member of the tumor necrosis factor receptor (TNFR) family expressed in osteoclast precursors, and RANK-RANK ligand (RANKL) signaling is a key system for differentiation, activation and survival of osteoclasts. Here, we report the identification of a novel alternative splicing variant of mouse RANK gene (vRANK) that contains a new intervening exon between exon 1 and exon 2 of mouse full-length RANK (fRANK) mRNA. Since this novel exon contains the stop codon, vRANK encodes truncated amino acids that have a portion of the signal peptide of fRANK and an additional 19 amino acids that show no homology to previously reported domains. By transient transfection studies with vRANK-GFP and -Flag expressing constructs, vRANK was found localized mostly in the cytoplasm and partly in the cell membrane, but was not secreted into the culture supernatant. Under the stimulation of various factors, the expression of vRANK mRNA was almost parallel to that of fRANK in RAW264.7 cells not treated with M-CSF. Overexpression of vRANK, on the other hand, decreased TRACP (a marker of osteoclasts) mRNA expression as well as the number of TRACP-positive multinucleated giant cells. While the mRNA expression levels of NFATc1 (a master transcriptional factor of the osteoclast differentiation program) were not affected, apoptotic cells increased significantly in vRAN K-transfected cells treated with sRANKL. Taken together, these results suggest that vRANK is a novel osteoclast suppressor that reduces the number of RANKL-induced mature osteoclasts mainly by negating the anti-apoptotic effect of RANKL.
Satomi Mukai, Riko Kitazawa, Junko Ishii, Takeshi Kondo, Akihiro Hakozaki, Keisuke Horiuchi, Ryuma Haraguch, Kiyoshi Mori, Sohei Kitazawa

2677 related Products with: Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB.

100ug100ug100.00 ug 5 G100ul100 0.1ml25 TESTS100ug100 TESTS

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#19540841   2009/06/21 To Up

Regulation of nuclear factor of activated T cells (NFAT) in vascular endothelial cells.

Proteins of the NFAT family (nuclear factor of activated T cells) are Ca(2+)-sensitive transcription factors, which are involved in hypertrophic cardiovascular remodeling. Activation and nuclear translocation is mediated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin (CaN). We identified Ca(2+) signals that induced nuclear translocation of NFAT in cultured calf pulmonary artery endothelial (CPAE) cells using confocal fluorescence microscopy to measure simultaneously [Ca(2+)](i) and subcellular localization of NFAT-GFP (isoforms NFATc1 and NFATc3). The vasoactive agonists ATP (5 microM) or bradykinin (20 microM) in the presence of 2 mM extracellular Ca(2+) induced Ca(2+) release from the endoplasmic reticulum (ER) and activated capacitative Ca(2+) entry (CCE), which caused robust translocation of NFAT to the nucleus. This effect was sensitive to the CaN-inhibitor cyclosporin A (1 microM). Influx of extracellular Ca(2+) via CCE, but not ER Ca(2+) release was identified as the activating Ca(2+) source. NFAT was also activated by Ca(2+) influx induced by cell swelling, reverse mode Na/Ca exchange or ionomycin treatment. NFAT regulation was isoform-specific. Whereas activation of NFATc1-GFP by ATP resulted in persistent nuclear localization, NFATc3-GFP was only transiently imported into the nucleus, followed by rapid export back to the cytoplasm. Inhibition of nuclear kinases, which mediate export of NFAT via phosphorylation, or direct block of nuclear export (Leptomycin B) resulted in stable nuclear localization of NFATc3. These data demonstrate that extracellular Ca(2+) entry mediates NFAT activation. Furthermore, the regulation of nuclear localization of NFAT is isoform-specific and dependent on nuclear export processes.
Andreas Rinne, Kathrin Banach, Lothar A Blatter

2787 related Products with: Regulation of nuclear factor of activated T cells (NFAT) in vascular endothelial cells.

96 wells1.00 flask5 x 50 ug1.00 flask10 ug100 ug/vial50 ug96 tests1.00 flask1 mg1.00 flask

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#19463978   2009/05/20 To Up

NFATc1 mediates HDAC-dependent transcriptional repression of osteocalcin expression during osteoblast differentiation.

We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter, possibly in an HDAC-dependent manner.
Min-Kyung Choo, Hyeonju Yeo, Majd Zayzafoon

2615 related Products with: NFATc1 mediates HDAC-dependent transcriptional repression of osteocalcin expression during osteoblast differentiation.

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