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NOTCH2 protein, human
NOTCH2 protein, human
The NOTCH2 protein is a member of the Notch family of transmembrane receptors, which play crucial roles in cell-cell communication and regulation of cellular differentiation.
This protein is involved in a variety of developmental and homeostatic processes, including stem cell maintenance, cell fate determination, and tissue patterning.
Mutations or dysregulation of the NOTCH2 gene have been implicated in several human diseases, such as Alagille syndrome, a multisystemic disorder affecting the liver, heart, skeleton, and other organs.
Researchers studying the NOTCH2 protein may utilize a variety of experimental protocols and reagents to investigate its structure, function, and interactions.
Optimizing these research approaches and accessing the latest findings can be facilitated by tools like PubCompare.AI, which leverages the power of AI-driven comparisons to help locate the best protocols and products from the scientific literature, preprints, and patents.
Expereince the future of NOTCH2 protein research today with PubCompare.AI's intuitive platform.
This protein is involved in a variety of developmental and homeostatic processes, including stem cell maintenance, cell fate determination, and tissue patterning.
Mutations or dysregulation of the NOTCH2 gene have been implicated in several human diseases, such as Alagille syndrome, a multisystemic disorder affecting the liver, heart, skeleton, and other organs.
Researchers studying the NOTCH2 protein may utilize a variety of experimental protocols and reagents to investigate its structure, function, and interactions.
Optimizing these research approaches and accessing the latest findings can be facilitated by tools like PubCompare.AI, which leverages the power of AI-driven comparisons to help locate the best protocols and products from the scientific literature, preprints, and patents.
Expereince the future of NOTCH2 protein research today with PubCompare.AI's intuitive platform.
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For lineage analysis in the intestine, four week-old mice, including control mice, received one single intra-peritoneal injection of tamoxifen (ICN) (50 or 5 mg/Kg of mouse body weight) and were analyzed at the indicated chase time points. To confirm the absence of leakiness of the new transgenic lines, we analyzed Notch(1–2)-CreERT2SAT/+; R26R/+ and N(1–2)-CreERT2SAT/+; R26mTmG/+ mice without tamoxifen administration and found no LacZ nor GFP staining in these control mice. For expression analysis in other organs we used a single intra-peritoneal injection of 4-Hydroxytamoxifen (4-OHT) (50 mg/kg of mouse body weight).
Pharmacological inhibition of Notch signaling in Hes1-EmGFPSAT mice was achieved by intraperitoneal administration of the γ-secretase inhibitor dibenzazepine (DBZ: 20 µM/kg body weight in 0.5% HPMC, 0.1% w/v NP40 in water; Calbiochem). Adult mice were injected with DBZ for 4 consecutive days and analyzed 3–4 days after the last injection.
Pharmacological inhibition of Notch signaling in Hes1-EmGFPSAT mice was achieved by intraperitoneal administration of the γ-secretase inhibitor dibenzazepine (DBZ: 20 µM/kg body weight in 0.5% HPMC, 0.1% w/v NP40 in water; Calbiochem). Adult mice were injected with DBZ for 4 consecutive days and analyzed 3–4 days after the last injection.
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4,17 beta-dihydroxy-4-androstene-3-one
Adult
Animals, Transgenic
Body Weight
dibenzazepine
hydroxytamoxifen
Injections, Intraperitoneal
Intestines
LacZ Genes
Mice, Laboratory
NOTCH2 protein, human
Psychological Inhibition
Secretase
Tamoxifen
We used two sample t tests to compare mean baseline values of continuous variables in people who developed diabetes and those who did not. Where appropriate, we log transformed variables and present geometric means and approximate standard deviations. We used the χ2 test to compare categorical variables. We assessed the association of each genotype with risk of diabetes by logistic regression analysis and summarised the data by odds ratios and 95% confidence intervals. We used published regression coefficients to calculate the Cambridge type 2 diabetes risk score and Framingham offspring study type 2 diabetes risk score for each participant.7 (link)
8 (link) In addition, we calculated two genetic scores. In the first, we assigned each person a score based simply on the number of risk alleles carried. Thus for CDKAL1, CDC123/CAMK1D, FTO, HNF1A, IGFBP2, KCNJ11, NOTCH2, TCF2, TCF7L2, TSPAN8/LRG5, and VEGFA, we coded genotypes “0” for common allele homozygotes,11 (link) “1” for heterozygotes, and “2” for rare allele homozygotes,22 (link) and for ADAMTS9, BCL11A, CALPN10, CDKN2A/2B, HHEX, JAZF1, PPARG, SLC30A8, and THADA, coding was “2” for common allele homozygotes and “0” for rare allele homozygotes,11 (link) as the rare allele is reported to be protective (see web table B). In the second score, we calculated a genetic risk function by using weights derived from the risk coefficient for each gene based on odds ratios reported in previous meta-analyses (web table A).15 (link)
16 (link)
18 (link)
32 (link) Risk estimates for each allele were available for 18 genes, and we multiplied these coefficients by 0, 1, or 2 according to the number of risk alleles carried by each person. Where effect estimates were reported for carriage of either one or two copies of each risk allele as a single group (CALPN10 and HNF1A), we multiplied risk coefficients by a score of 0 or 1. We assumed genetic and clinical variables to be independent and added the weighted genetic score to each of the risk algorithms to provide a combined phenotypic and genetic score.
We assessed discrimination with the detection rate, which is equivalent to sensitivity and defined as the proportion of all cases detected for a pre-specified false positive rate, as well as the area under the receiver operating characteristics curve. We assessed the calibration of the Cambridge risk score and Framingham offspring risk score in the estimation of the absolute risk of type 2 diabetes by comparing the difference between observed and expected event rates in different categories of risk with the Hosmer-Lemeshow test, with Akaike’s information criterion and the likelihood ratio test as global measures of model fit.33 (link) We used the net reclassification improvement measure to assess the extent to which adding the genetic variables reassigned people to risk categories that better reflected their final outcome.34 (link)
8 (link) In addition, we calculated two genetic scores. In the first, we assigned each person a score based simply on the number of risk alleles carried. Thus for CDKAL1, CDC123/CAMK1D, FTO, HNF1A, IGFBP2, KCNJ11, NOTCH2, TCF2, TCF7L2, TSPAN8/LRG5, and VEGFA, we coded genotypes “0” for common allele homozygotes,11 (link) “1” for heterozygotes, and “2” for rare allele homozygotes,22 (link) and for ADAMTS9, BCL11A, CALPN10, CDKN2A/2B, HHEX, JAZF1, PPARG, SLC30A8, and THADA, coding was “2” for common allele homozygotes and “0” for rare allele homozygotes,11 (link) as the rare allele is reported to be protective (see web table B). In the second score, we calculated a genetic risk function by using weights derived from the risk coefficient for each gene based on odds ratios reported in previous meta-analyses (web table A).15 (link)
16 (link)
18 (link)
32 (link) Risk estimates for each allele were available for 18 genes, and we multiplied these coefficients by 0, 1, or 2 according to the number of risk alleles carried by each person. Where effect estimates were reported for carriage of either one or two copies of each risk allele as a single group (CALPN10 and HNF1A), we multiplied risk coefficients by a score of 0 or 1. We assumed genetic and clinical variables to be independent and added the weighted genetic score to each of the risk algorithms to provide a combined phenotypic and genetic score.
We assessed discrimination with the detection rate, which is equivalent to sensitivity and defined as the proportion of all cases detected for a pre-specified false positive rate, as well as the area under the receiver operating characteristics curve. We assessed the calibration of the Cambridge risk score and Framingham offspring risk score in the estimation of the absolute risk of type 2 diabetes by comparing the difference between observed and expected event rates in different categories of risk with the Hosmer-Lemeshow test, with Akaike’s information criterion and the likelihood ratio test as global measures of model fit.33 (link) We used the net reclassification improvement measure to assess the extent to which adding the genetic variables reassigned people to risk categories that better reflected their final outcome.34 (link)
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Alleles
BCL11A protein, human
CDKAL1 protein, human
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Discrimination, Psychology
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Genes, vif
Genotype
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NOTCH2 protein, human
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zinc transporter 8, human
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Malignant Neoplasms
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Oncogenes
One-Step dentin bonding system
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RNA-Seq
Tissues
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NOTCH2 protein, human
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Most recents protocols related to «NOTCH2 protein, human»
Cell lysates of ALDH−, ALDH+, and CD44+/CD24− cells following treatment with vehicle and treatment (ASR490, DAPT, MG132, CHX, or CQ) for prescribed doses and time points, were prepared with RIPA buffer (Thermo Scientific, Rockford, IL, United States) per the manufacturer’s protocol. Western blotting was performed using specific antibodies against Notch1 (CST, #3608), HES1 (Sigma, #SAB2108472), Hey1 (Proteintech, #19929-1-AP), NFκB p65 (CST, #8242), Bcl-2 (CST, #15071), Bcl-xL (CST, #2764), Vimentin (CST, #46173), Slug (CST, #9585), E-Cadherin (CST, #3195), β-catenin (CST, #8480), Ubiquitin (CST, #3933), Cleaved-PARP (CST, #5625), Cleaved-caspase-9 (CST, #20750), BAX (CST, #41162), Notch2 (CST, #D76A6), Lamp1 (CST, #9091), and LC3B (Proteintech, #14600-1-AP). β-Actin (CST, #4970) was used as the loading control. Protein bands were visualized using the Bio-Rad ChemiDocTM imaging system. For IP experiments, protein samples were immunoprecipitated with Notch1 antibody as per the protocol described elsewhere (Chandrasekaran et al., 2020 (link)), and Western blots were performed with ubiquitin antibody.
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1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol
Actins
Antibodies
BCL2 protein, human
beta-Catenin
Buffers
Caspase 9
CD44 protein, human
CDH1 protein, human
Cells
Immunoglobulins
lysosomal-associated membrane protein 1, human
MG 132
NOTCH2 protein, human
Proteins
Radioimmunoprecipitation Assay
Slugs
Transcription Factor RelA
Ubiquitin
Vimentin
Western Blot
Brain samples that were conserved in RNA later at -80 ºC since eradication were used to assess the expression of the genes: CXCL-1,IL-1β, and Notch2 using the CDKn1-a as a housekeeping gene for normalization. Primers were synthesized by Invitrogen (Carlsbad, CA, USA)5 (link). RNA was extracted from the samples with RNeasy extraction kit according to manufacturer recommended protocol of (Qiagen GMbH, Germany). Purity and quantity of the RNA samples were assessed using Thermo Scientific NanoDrop 2000 to assure that concentrations were pure enough to conduct RT-PCR. cDNA was synthesized with an adjusted input of 1 µg of RNA extract using Reverse transcription kit (Thermo Fisher Scientific, RevertAid RT Reverse transcription Kit). RT-qPCR was performed using Master mix (SensiFAST ® SYBER No-ROX Kit) in RotorGene-6000 system (Corbett Robotics Australia) following the manufacturer protocol. For qPCR data analysis, Rotor-Gene Q series software (v.1.7) was used for a comparative quantification analysis. Relative gene expressions were calculated with the improved 2-ΔΔCT method that directly uses the CT values obtained from the real time qPCR system67 .
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Brain
DNA, Complementary
Gene Expression
Genes
Genes, Housekeeping
Interleukin-1 beta
NOTCH2 protein, human
Oligonucleotide Primers
Reverse Transcriptase Polymerase Chain Reaction
Reverse Transcription
Sequence search-based modeling of Fibulin2 was performed using the SWISS-MODEL database (swissmodel.expasy.org) based on the structure of Dll1 (PDB ID: 4xbm.2. A, 31.44% shared identity). The SWISS-MODEL-predicted structures were analyzed for the QMEAN Z score, which included the cumulative Z score of the Cβ, all atoms, solvation and torsion values. The Notch2 domain structure was downloaded from the RCSB PDB database (www1.rcsb.org). The initial docking between the Fibulin2 and Notch2 structures was performed with Z-Dock (3.0.2). Then, model screening was conducted using PDBePISA to identify all conformations (www.ebi.ac.uk/msd-srv/ssm/ ).
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NOTCH2 protein, human
Rumex
Bone samples were fixed with 4% paraformaldehyde for 24 h, decalcified with 15% EDTA for 2 weeks, processed into paraffin sections at a thickness of 4 μm, and subjected to HE and Masson’s trichrome staining. Immunohistochemistry to detect Fibulin2, Notch2, and Runx2 on the sections was performed using primary antibodies against Fibulin2 (Abcam, ab234993, 1:250), Notch2 (CST, 57325, 1:100), and Runx2 (Abcam, ab192256, 1:250). The stained sections were scanned under a microscope (Leica, Germany).
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Antibodies
Bones
Edetic Acid
Immunohistochemistry
Microscopy
NOTCH2 protein, human
Paraffin
paraform
RUNX2 protein, human
shRNA against Fibulin2 was used to establish sh-FBLN2 in a GV493 lentiviral vector. Full-length FBLN2 was cloned into a GV640 lentiviral vector. All lentiviruses and respective negative controls were synthesized by GeneChem (Shanghai, China). BMSCs were cultured in 25 cm2 monolayers in culture medium to a density of approximately 5000 cells per cm2. The cells were transfected at a multiplicity of infection of 10 with lentiviral particles expressing shRNA directed against the Fibulin2 transcript (Shanghai GeneChem Co.), nontargeting control shRNA (scramble), or an unloaded control (vehicle) for 24 h. Then, the cell monolayers were washed in PBS, cultured for an additional 24 h, and selected for 2 days with 1 μg/mL puromycin (Beyotime, China).
Coimmunoprecipitation. Coimmunoprecipitation was performed as previously described29 (link). BMSCs were transfected with lentivirus encoding 3×Flag-FBLN2. The transfected cells were cultured for 36 to 48 h. For immunoprecipitation of 3×Flag-tagged Fibulin2 and Notch2, the cells were lysed in lysis buffer (Thermo Fisher, USA). The whole cell lysates were precleared with rProtein G Sepharose (Thermo Fisher, USA) and 2 µg of control IgG (Abcam, ab172730) at 4 °C for 1 h. The supernatants were collected and incubated at 4 °C for 4 h with either anti-3×Flag antibody (CST, 14793S), anti-Notch2 antibody (CST, 7532S), or control IgG (Abcam, ab172730). Then, the immune complexes were collected after incubation for 1 h at 4 °C with rProtein G Sepharose (Thermo Fisher, USA). After three washes in wash buffer, the immunoprecipitates were boiled in 2X loading buffer (Beijing Biomed Gene Technology Co.) for 10 min and subjected to immunoblotting.
Coimmunoprecipitation. Coimmunoprecipitation was performed as previously described29 (link). BMSCs were transfected with lentivirus encoding 3×Flag-FBLN2. The transfected cells were cultured for 36 to 48 h. For immunoprecipitation of 3×Flag-tagged Fibulin2 and Notch2, the cells were lysed in lysis buffer (Thermo Fisher, USA). The whole cell lysates were precleared with rProtein G Sepharose (Thermo Fisher, USA) and 2 µg of control IgG (Abcam, ab172730) at 4 °C for 1 h. The supernatants were collected and incubated at 4 °C for 4 h with either anti-3×Flag antibody (CST, 14793S), anti-Notch2 antibody (CST, 7532S), or control IgG (Abcam, ab172730). Then, the immune complexes were collected after incubation for 1 h at 4 °C with rProtein G Sepharose (Thermo Fisher, USA). After three washes in wash buffer, the immunoprecipitates were boiled in 2X loading buffer (Beijing Biomed Gene Technology Co.) for 10 min and subjected to immunoblotting.
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Antibodies, Anti-Idiotypic
Buffers
Cell Culture Techniques
Cells
Cloning Vectors
Co-Immunoprecipitation
Complex, Immune
FBLN2 protein, human
Genes
Immunoglobulins
Immunoprecipitation
Infection
Lentivirus
NOTCH2 protein, human
Puromycin
Sepharose
Short Hairpin RNA
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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Notch2 is a transmembrane protein that functions as a receptor in the Notch signaling pathway. It plays a role in cell-cell communication and is involved in regulating various cellular processes such as cell fate determination, differentiation, and proliferation.
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The RNeasy Mini Kit is a laboratory equipment designed for the purification of total RNA from a variety of sample types, including animal cells, tissues, and other biological materials. The kit utilizes a silica-based membrane technology to selectively bind and isolate RNA molecules, allowing for efficient extraction and recovery of high-quality RNA.
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Ab8926 is a monoclonal antibody that recognizes the human CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells. This antibody can be used for the identification and characterization of cells expressing CD45.
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The Dual-Luciferase Reporter Assay System is a laboratory tool designed to measure and compare the activity of two different luciferase reporter genes simultaneously. The system provides a quantitative method for analyzing gene expression and regulation in transfected or transduced cells.
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Notch1 is a cell surface receptor that plays a crucial role in cell-cell communication and signal transduction. It is involved in the Notch signaling pathway, which regulates various cellular processes, including cell fate determination, differentiation, and proliferation. Notch1 contains an extracellular domain that can bind to ligands on neighboring cells, leading to the activation of the intracellular domain and the subsequent transduction of the Notch signal within the cell.
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β-actin is a cytoskeletal protein that is ubiquitously expressed in eukaryotic cells. It is a component of the microfilament system and plays a crucial role in various cellular processes, such as cell motility, maintenance of cell shape, and intracellular trafficking.
More about "NOTCH2 protein, human"
The NOTCH2 protein is a crucial member of the Notch family of transmembrane receptors, which play pivotal roles in cell-cell communication and regulation of cellular differentiation.
This protein is involved in a variety of essential developmental and homeostatic processes, including stem cell maintenance, cell fate determination, and tissue patterning.
Mutations or dysregulation of the NOTCH2 gene have been implicated in several human diseases, such as Alagille syndrome, a multisystemic disorder affecting the liver, heart, skeleton, and other organs.
Researchers studying the NOTCH2 protein may utilize a variety of experimental protocols and reagents, such as Lipofectamine 2000 for transfection, TRIzol reagent for RNA extraction, and the Dual-Luciferase Reporter Assay System for gene expression analysis.
The RNeasy Mini Kit is also a commonly used tool for high-quality RNA purification.
To optimize these research approaches and access the latest findings, researchers can leverge the power of AI-driven comparison tools like PubCompare.AI.
This platform helps locate the best protocols and products from the scientific literature, preprints, and patents, facilitating efficient and reproducible NOTCH2 protein research.
Expereince the future of NOTCH2 protein research today with PubCompare.AI's intuitive platform, which empowers researchers to streamline their workflows, enhance their findings, and explore the latest advancements in this important field of study.
This protein is involved in a variety of essential developmental and homeostatic processes, including stem cell maintenance, cell fate determination, and tissue patterning.
Mutations or dysregulation of the NOTCH2 gene have been implicated in several human diseases, such as Alagille syndrome, a multisystemic disorder affecting the liver, heart, skeleton, and other organs.
Researchers studying the NOTCH2 protein may utilize a variety of experimental protocols and reagents, such as Lipofectamine 2000 for transfection, TRIzol reagent for RNA extraction, and the Dual-Luciferase Reporter Assay System for gene expression analysis.
The RNeasy Mini Kit is also a commonly used tool for high-quality RNA purification.
To optimize these research approaches and access the latest findings, researchers can leverge the power of AI-driven comparison tools like PubCompare.AI.
This platform helps locate the best protocols and products from the scientific literature, preprints, and patents, facilitating efficient and reproducible NOTCH2 protein research.
Expereince the future of NOTCH2 protein research today with PubCompare.AI's intuitive platform, which empowers researchers to streamline their workflows, enhance their findings, and explore the latest advancements in this important field of study.