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The following MAPmates™ should not be plexed together: -MAPmates™ that require a different assay buffer -Phospho-specific and total MAPmate™ pairs, e.g. total GSK3β and GSK3β (Ser 9) -PanTyr and site-specific MAPmates™, e.g. Phospho-EGF Receptor and phospho-STAT1 (Tyr701) -More than 1 phospho-MAPmate™ for a single target (Akt, STAT3) -GAPDH and β-Tubulin cannot be plexed with kits or MAPmates™ containing panTyr
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48-602MAG
Buffer Detection Kit for Magnetic Beads
1 Kit
Space Saver Option Customers purchasing multiple kits may choose to save storage space by eliminating the kit packaging and receiving their multiplex assay components in plastic bags for more compact storage.
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To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor (HGFR)-expressing cells in active ulcerative colitis (UC).On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected. After preparing tissue microarrays and blood smears HGFR, caudal type homeobox 2 (CDX2), prominin-1 (CD133) and Musashi-1 conventional and double fluorescent immunolabelings were performed. Immunostained samples were digitalized using high-resolution Mirax Desk instrument, and analyzed with the Mirax TMA Module software. For semiquantitative counting of immunopositive lamina propria (LP) cells 5 fields of view were counted at magnification × 200 in each sample core, then mean ± SD were determined. In case of peripheral blood smears, 30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells (mean ± SD) was determined. Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected. Gene expression analysis of HGFR, CDX2, CD133, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), Musashi-1 and cytokeratin 20 (CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction (RT-PCR).By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR, higher number of HGFR (blood: 6.7 ± 1.22 vs 38.5 ± 3.18; LP: 2.25 ± 0.85 vs 9.22 ± 0.65; P less than 0.05), CDX2 (blood: 0 vs 0.94 ± 0.64; LP: 0.75 ± 0.55 vs 2.11 ± 0.75; P less than 0.05), CD133 (blood: 1.1 ± 0.72 vs 8.3 ± 1.08; LP: 11.1 ± 0.85 vs 26.28 ± 1.71; P less than 0.05) and Musashi-1 (blood and LP: 0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls. HGFR/CDX2 (blood: 0 vs 1 ± 0.59; LP: 0.8 ± 0.69 vs 2.06 ± 0.72, P less than 0.05) and Musashi-1/CDX2 (blood and LP: 0 vs scattered) co-expressions were found in blood and lamina propria of UC samples. HGFR/CD133 and CD133/CDX2 co-expressions appeared only in UC lamina propria samples. CDX2, Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression.In active UC, a portion of circulating HGFR-expressing cells are committed to the epithelial lineage, and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.
Previous functional studies have demonstrated that muscle intracellular pH regulation is mediated by sodium-coupled bicarbonate transport, Na+/H+ exchange, and Cl-/bicarbonate exchange. We report the cloning, sequence analysis, tissue distribution, genomic organization, and functional analysis of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC3, from human skeletal muscle. mNBC3 encodes a 1214-residue polypeptide with 12 putative membrane-spanning domains. The approximately 7.8-kilobase transcript is expressed uniquely in skeletal muscle and heart. The NBC3 gene (SLC4A7) spans approximately 80 kb and is composed of 25 coding exons and 24 introns that are flanked by typical splice donor and acceptor sequences. Expression of mNBC3 cRNA in Xenopus laevis oocytes demonstrated that the protein encodes a novel stilbene-insensitive 5-(N-ethyl-N-isopropyl)-amiloride-inhibitable sodium bicarbonate cotransporter.
Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.
Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.
FOXP2 mutation causes a severe inherited speech and language defect, while the related transcription factors FOXP1, FOXP3 and FOXP4 are implicated in cancer. FOXP2 mRNA and protein expression were characterised in normal human tissues, haematological cell lines and multiple myeloma (MM) patients' samples. FOXP2 mRNA and protein were absent in mononuclear cells from different anatomical sites, lineages and stages of differentiation. However, FOXP2 mRNA and protein was detected in several lymphoma (8/20) and all MM-derived cell lines (n = 4). FOXP2 mRNA was expressed in bone marrow samples from 96% of MM patients (24/25), 66.7% of patients with the pre-neoplastic plasma cell proliferation monoclonal gammopathy of undetermined significance (MGUS) (6/9), but not in reactive plasma cells. The frequency of FOXP2 protein expression in CD138(+) plasma cells was significantly higher in MGUS (P = 0.0005; mean 46.4%) and MM patients (P < or = 0.0001; mean 57.3%) than in reactive marrows (mean 2.5%). FOXP2 (>10% nuclear positivity) was detectable in 90.2% of MM (55/61) and 90.9% of MGUS (10/11) patients, showing more frequent expression than CD56 and labelling 75% of CD56-negative MM (9/12). FOXP2 represents the first transcription factor whose expression consistently differentiates normal and abnormal plasma cells and FOXP2 target genes are implicated in MM pathogenesis.
Metabolic disorders induced by high-fat feeding in rodents evoke some, if not all, of the features of human metabolic syndrome. The occurrence and severity of metabolic disorders, however, varies according to rodent species, and even strain, as well as the diet. Therefore, in the present study, we investigated the long-term obesogenic and diabetogenic effects of three high-fat diets differing by their fat/carbohydrate ratios. Sprague-Dawley rats were fed a control high-carbohydrate and low-fat diet [HCD; 3:16:6 ratio of fat/carbohydrate/protein; 15.48 kJ/g (3.7 kcal/g)], a high-fat and medium-carbohydrate diet [HFD1; 53:30:17 ratio of fat/carbohydrate/protein; 19.66 kJ/g (4.7 kcal/g)], a very-high-fat and low-carbohydrate diet [HFD2; 67:9:24 ratio of fat/carbohydrate/protein; 21.76 kJ/g (5.2 kcal/g)] or a very-high-fat and carbohydrate-free diet [HFD3; 75:0:25 ratio of fat/carbohydrate/protein; 24.69 kJ/g (5.9 kcal/g)] for 10 weeks. Compared with the control diet (HCD), rats fed with high-fat combined with more (HFD1) or less (HFD2) carbohydrate exhibited higher BMI (body mass index; +13 and +10% respectively; P0.05) and abdominal fat (+70% in both HFD1 and HFD2; P0.05), higher plasma leptin (+130 and +135% respectively; P0.05), lower plasma adiponectin levels (-23 and -30% respectively; P0.05) and impaired glucose tolerance. Only the HFD1 group had insulin resistance. By contrast, a very-high-fat diet devoid of carbohydrate (HFD3) led to impaired glucose tolerance, insulin resistance and hypoadiponectinaemia (-50%; P0.05), whereas BMI, adiposity and plasma leptin did not differ from respective values in animals fed the control diet. We conclude that increasing the fat-to-carbohydrate ratio to the uppermost (i.e. carbohydrate-free) in a high-fat diet prevents the development of obesity, but not the prediabetic state (i.e. altered glucose tolerance and insulin sensitivity).
We report here the isolation, characterization on genomic structure and expression of the D. melanogaster homolog of human parkin. The 2,122 bp parkin gene sequence contains six exons that form a 1,449 bp transcript encoding a protein of 482 amino acids. 151 bp of 5' and 112 bp of 3' untranslated regions were identified by a combination of 5'-RACE/primer extension and 3'-RACE, respectively. The 5' UTR contains three transcription initiation sites. Neither a classical TATA nor a CAAT box was found in the putative promoter sequence. However, binding sites for AhR-Arnt, AP4, NF1 and GATA transcription factors were identified. Transient transfection analysis of the 5' UTR confirmed its promoter activity in HEK 293 cells and SH-SY5Y neuronal cells using a dual luciferase reporting system. The amino acid sequence of D. melanogaster Parkin exhibits 42%, 43% and 43% identity to that of human, mouse and rat, respectively, representing a 54 kDa protein band via western blot analysis. It shows a high degree of conservation in the Ubiquitin-like domain at the N-terminus (34%), the In-Between RING finger domains (IBR, 65-69%), and the RING finger domains at the C-terminus (56-57%). The expression pattern of D. melanogaster parkin varies during the developmental stages, with the highest expression in the adult stage as measured by competitive RT-PCR. From immunostainings of the embryo, D. melanogaster parkin was expressed slightly higher in the central nervous system (brain and nerve cord) during the late embryonic stage.