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LEF1 protein, human
LEF1 protein, human
LEF1 (Lymphoid Enhancer Binding Factor 1) is a transcription factor that plays a key role in the Wnt signaling pathway and is involved in the regulation of cell proliferation, differentiation, and development.
It is expressed in various tissues, including the lymphoid system, where it is essential for T-cell maturation and B-cell development.
LEF1 has been implicated in several disease processes, such as cancer, autoimmune disorders, and neurological conditions.
Researchers studying the LEF1 protein can leverage the PubCompare.ai tool to optimize their research workflow, identify the best methodologies and products from literature, preprints, and patents, and enhance reproducibility of their studies.
It is expressed in various tissues, including the lymphoid system, where it is essential for T-cell maturation and B-cell development.
LEF1 has been implicated in several disease processes, such as cancer, autoimmune disorders, and neurological conditions.
Researchers studying the LEF1 protein can leverage the PubCompare.ai tool to optimize their research workflow, identify the best methodologies and products from literature, preprints, and patents, and enhance reproducibility of their studies.
Most cited protocols related to «LEF1 protein, human»
Antibodies
Body Weight
Bromodeoxyuridine
Embryo
Eosin
Ethanol
Euthanasia
Exons
Fixatives
Immunofluorescence
In Situ Hybridization
LEF1 protein, human
Orange G
Paraffin Embedding
RNA, Messenger
Sucrose
Tyrosine 3-Monooxygenase
WNT1 protein, human
Animals
LEF1 protein, human
Mice, Laboratory
Mice, Transgenic
RAG-1 Gene
TCF7 protein, human
VAV1 protein, human
For immunoblot analyses, known quantities of recombinant human keratins were electrophoresed, transferred to nitrocellulose, and the blots were incubated with primary antisera diluted in blocking buffer (Tris-buffered saline with 0.5% Tween 20 and 5% powdered milk). Bound primary antibodies were revealed by alkaline phosphatase–conjugated secondary antibodies as recommended by the manufacturer (Bio-Rad Laboratories). The primary antisera for K17 and K6 were prepared as described below. Immunohistochemical analyses were performed on 5-μm sections prepared from either paraffin-embedded or fresh-frozen tissues. The sections were first reacted with the primary antisera and then revealed with either a peroxidase-based reaction (Kirkegaard and Perry Laboratories, Gaithersburg, MD) or by indirect immunofluorescence (Jackson Immunological Reagents, West Grove, PA). In addition to the antisera mentioned above, other primary antisera used in these studies were: a guinea pig anti-K8/18 purchased from ARP laboratories (Belmont, MA); a mouse monoclonal anti-K10 (clone K8.60) from Sigma Chemical Co. ; a mouse monoclonal anti-K14 (LL001) donated by I. Leigh (London Hospital Medical College, London, UK) (Purkis et al., 1990 (link)); and a rabbit anti–lef-1 donated by R. Grosschedl (van Genderen et al., 1994 (link)). Double-immunofluorescence staining using primary antibodies from the same species (rabbit anti-K17 and rabbit anti–lef-1) was performed as described (Lewis-Carl et al., 1993 (link)).
Alkaline Phosphatase
Antibodies
Antibody Specificity
Buffers
Cavia porcellus
Clone Cells
Cytokeratin
Fluorescent Antibody Technique
Freezing
Homo sapiens
Immune Sera
Immunoblotting
Indirect Immunofluorescence
LEF1 protein, human
Mice, House
Milk, Cow's
Nitrocellulose
Paraffin Embedding
Peroxidase
Rabbits
Saline Solution
Tissues
Tween 20
Gene Expression
Genotype
LEF1 protein, human
Lymphoma
Mus
RAG-1 Gene
Student
TCF7 protein, human
Total RNA was extracted from the sorted PD-1+CXCR5+ cells from Tcf7−/−Lef1−/− or control mice, and two biological replicates were obtained for each genotype. cDNA synthesis and amplification were performed using SMARTer Ultra Low Input RNA Kit (Clontech) starting with 10 ng of total RNA per sample following manufacturer’s instructions. cDNA was fragmented with Q800R sonicator (Qsonica) and used as input for NEBNext Ultra DNA Library Preparation Kit (NEB). Libraries were sequenced on Illumina’s HiSeq2000 in single read mode with the read length of 50 nucleotides producing 60–70 million reads per sample. Sequence data in fastq format were generated using CASAVA 1.8.2 processing pipeline from Illumina.
The sequencing quality of RNA-Seq libraries was assessed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ , v0.10.1). Because of biased GC content in the 5’ end, the first 12 bases of each read in all 4 samples were trimmed off. RNA-Seq data reproducibility was evaluated by computing Pearson’s correlation of FPKM values for all genes between biological replicates. The Pearson’s correlation coefficient between the two biological replicates was 0.937 for the control samples and 0.986 for the Tcf7−/−Lef1−/− samples, indicating strong reproducibility.
The RNA-Seq libraries were then processed by RSEM (v1.2.19) to estimate expression levels of all genes. The expression level of a gene is expressed as a gene-level FPKM (Fragments Per Kilobase of transcripts per Million mapped reads) value. EBSeq (v1.5.4), as an integral component of RSEM package, was used to identify differentially expressed genes. UCSC genes for mouse mm9 from iGenome (http://support.illumina.com/sequencing/sequencing_software/igenome.html ) were used for gene annotation. The RNA-seq data are deposited at the Gene Expression Omnibus under accession number GSE66781.
The sequencing quality of RNA-Seq libraries was assessed by FastQC (
The RNA-Seq libraries were then processed by RSEM (v1.2.19) to estimate expression levels of all genes. The expression level of a gene is expressed as a gene-level FPKM (Fragments Per Kilobase of transcripts per Million mapped reads) value. EBSeq (v1.5.4), as an integral component of RSEM package, was used to identify differentially expressed genes. UCSC genes for mouse mm9 from iGenome (
Anabolism
Biopharmaceuticals
CXCR5 Receptors
DNA, Complementary
DNA Library
Gene Annotation
Gene Expression
Genes
Genotype
LEF1 protein, human
Mus
Nucleotides
RNA-Seq
TCF7 protein, human
Most recents protocols related to «LEF1 protein, human»
The following primary antibodies were used: anti-Krt14 (polyclonal chicken; 1:10,000; BioLegend), anti-GFP (green fluorescent protein; polyclonal rabbit; 1:1000; Abcam, RRID:AB_305564), anti-GFP (polyclonal goat; 1:1000; Abcam, RRID AB_305643), anti-GFP (polyclonal chicken; 1:500; Invitrogen, RRID:AB_2534023), anti-p63 (rabbit monoclonal; 1:1000; Abcam, RRID:AB_10971840), anti-Krt8 (rat monoclonal; 1:250; DSHB, RRID:AB_531826), anti-Entpd2 (rabbit; 1:4000; http://ectonucleotidases-ab.com , mN2-36Li6), anti-Gnat3 (goat polyclonal; 1:500; Novus Biologicals, NBP1-20926), anti-Snap25 (rabbit polyclonal; 1:500; Sigma-Aldrich, S9684), anti-Tas1r2 (rabbit; 1:500; Invitrogen, PA5-99935), anti-Lef1 (rabbit monoclonal; 1:100; Thermo Fisher Scientific, MA5-14966), anti-Sox2 (rabbit monoclonal; 1:200; Abcam ab92494).
The following secondary antibodies were used: anti-rabbit, anti-rat, anti-chicken, anti-goat conjugated to Alexa Fluor 488 (1:500; Jackson ImmunoResearch), to rhodamine Red-X (1:500; Jackson ImmunoResearch), or to Cy5 (1:1000; Jackson ImmunoResearch).
The following secondary antibodies were used: anti-rabbit, anti-rat, anti-chicken, anti-goat conjugated to Alexa Fluor 488 (1:500; Jackson ImmunoResearch), to rhodamine Red-X (1:500; Jackson ImmunoResearch), or to Cy5 (1:1000; Jackson ImmunoResearch).
alexa fluor 488
Antibodies
Biological Factors
Chickens
Goat
KRT8 protein, human
KRT14 protein, human
LEF1 protein, human
Novus
Rabbits
Rhodamine
SNAP25 protein, human
SOX2 protein, human
For the staining on frozen sections, tissues were harvested, directly embedded in O.C.T. (Tissue Tek) and flash-frozen for cryopreservation. For the following staining: Krt14, Krt8-YFP, Gnat3, Lef1, and Sox2, tissues were prefixed in 4% formaldehyde for 2 hours at room temperature, washed in phosphate-buffered saline (PBS), incubated overnight in 30% sucrose in PBS at 4°C, embedded in O.C.T. (Tissue Tek), and flash-frozen for cryopreservation. Samples were sectioned at 6μm using a M1860 cryostat (Leica Microsystems GmbH). Nonspecific antibody binding was blocked with 5% horse serum, 1% bovine serum albumin (BSA), and 0.2% Triton X-100 for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C in the same blocking buffer. Sections were rinsed three times in PBS and incubated with secondary antibodies for 1 hour at room temperature. Nuclei were stained with Hoechst (4 mM). Slides were mounted using Glycergel (Dako) supplemented with 2.5% DABCO (Sigma-Aldrich).
Cervical esophageal whole mount was prepared by cutting the cervical part of the esophagus, which is delimited by the cricoid cartilage proximally and the manubrium of the sternum distally. Tissue was incubated for 1 hour in 5 mM EDTA at 37°C on a rocking plate. Esophageal epithelium was separated from underlying tissue using forceps and then opened longitudinally with scissors. Tissue was fixed in 4% formaldehyde for 30 min at room temperature then rinsed in PBS. For staining, whole mount was blocked for 1 hour with 5% horse serum, 1% BSA, and 0.5% Triton X-100 in a 12-well plate. Whole mount was then stained with primary antibodies in blocking buffer (0.2% Triton X-100) overnight at 4°C. The following dilution has been used for the primary antibodies: anti-Krt14 (polyclonal chicken; 1:1000; BioLegend), anti-Krt8 (rat monoclonal; 1:1000; DSHB, RRID:AB_531826). Then, whole mount was incubated with secondary antibodies and Hoechst (4 mM) in blocking buffer (0.2% Triton X-100) overnight at 4°C using classical dilution of the secondary antibodies described below. Following each antibody incubation, whole mount was washed four times with 0.2% Tween 20 in PBS for 2 hours in total. Slides were mounted using Glycergel (Dako) supplemented with 2.5% Dabco (Sigma-Aldrich).
Cervical esophageal whole mount was prepared by cutting the cervical part of the esophagus, which is delimited by the cricoid cartilage proximally and the manubrium of the sternum distally. Tissue was incubated for 1 hour in 5 mM EDTA at 37°C on a rocking plate. Esophageal epithelium was separated from underlying tissue using forceps and then opened longitudinally with scissors. Tissue was fixed in 4% formaldehyde for 30 min at room temperature then rinsed in PBS. For staining, whole mount was blocked for 1 hour with 5% horse serum, 1% BSA, and 0.5% Triton X-100 in a 12-well plate. Whole mount was then stained with primary antibodies in blocking buffer (0.2% Triton X-100) overnight at 4°C. The following dilution has been used for the primary antibodies: anti-Krt14 (polyclonal chicken; 1:1000; BioLegend), anti-Krt8 (rat monoclonal; 1:1000; DSHB, RRID:AB_531826). Then, whole mount was incubated with secondary antibodies and Hoechst (4 mM) in blocking buffer (0.2% Triton X-100) overnight at 4°C using classical dilution of the secondary antibodies described below. Following each antibody incubation, whole mount was washed four times with 0.2% Tween 20 in PBS for 2 hours in total. Slides were mounted using Glycergel (Dako) supplemented with 2.5% Dabco (Sigma-Aldrich).
Antibodies
Cardiac Arrest
Cell Nucleus
Chickens
Cricoid Cartilage
Cryopreservation
Edetic Acid
Epithelium
Equus caballus
Esophagus
Forceps
Formaldehyde
Freezing
Frozen Sections
Immunoglobulins
KRT8 protein, human
KRT14 protein, human
LEF1 protein, human
Manubrium
Neck
Phosphates
Saline Solution
Serum
Serum Albumin, Bovine
SOX2 protein, human
Sucrose
Technique, Dilution
Tissues
triethylenediamine
Triton X-100
Tween 20
Skin biopsies were obtained using a 2 mm punch biopsy from target lesions chosen at both acral and non-acral sites. At baseline, biopsies were obtained from thirty recruited patients and were repeated at the end of the study period (24 weeks) from patients who completed the study. LEF1 expression was examined in paraffin-embedded skin tissue sections with immunohistochemistry (IHC) as described previously [5 (link)]. The expression of LEF1 was quantified as mean fluorescent intensity (MFI).
Biopsy
Immunohistochemistry
LEF1 protein, human
Paraffin Embedding
Patients
Skin
ES cells were washed with PBS, trypsinized, and collected by centrifugation. Whole cell lysates were prepared using RIPA cell lysis buffer (Sigma) containing 1:100 phosphatase inhibitor cocktail 2, phosphatase inhibitor cocktail 3 and protease inhibitor (Sigma). Samples were rotated for 30 min at 4 °C and spun at max speed for 10 min at 4 °C. The supernatant from samples was collected and protein concentration was determined by Bradford assay. Equal amounts of protein per sample were combined with Laemmli buffer, denatured for 5 min at 95 °C and subjected to SDS/PAGE separation, followed by immunoblotting. The following primary antibodies were used: rabbit anti-active β-Catenin (Cell Signaling Technology, 8814), mouse anti-total β-Catenin (BD Biosciences, 610154), rabbit monoclonal anti-Tcf1 (Cell Signaling Technology, 2203), rabbit monoclonal anti-Lef1 (Cell Signaling Technology, 2230). Mouse anti-β-ACTIN (Santa Cruz Biotechnology; sc-47778) was used as a load control. The antibodies used in this study can be found in Supplementary Table 1 . Protein quantification was performed with ImageJ software from n = 3 independent biological replicates (Fig. 1e ). Uncropped and unprocessed scans are provided in the Source data file (for Fig. 1e ) or in Supplementary Fig. 8 for blots presented in Supplementary figures.
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Actins
Antibodies
beta-Catenin
Biological Assay
Biopharmaceuticals
Buffers
Cells
Centrifugation
Embryonic Stem Cells
HNF1A protein, human
Laemmli buffer
LEF1 protein, human
Mus
Phosphoric Monoester Hydrolases
Protease Inhibitors
protein phosphatase inhibitor-2
Proteins
Rabbits
Radioimmunoprecipitation Assay
Radionuclide Imaging
SDS-PAGE
RNA was purified using TRIzol followed by digestion with RNase-free DNase (Qiagen). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription kit (ABI). Primer efficiencies were assessed by serial dilution. qRT–PCR reactions were performed in the QuantStudio 5 Real-Time PCR System using SYBR-Green Power master mix (ABI) with default cycling conditions; results were analyzed with QuantStudio 5 analysis software. All mRNA levels were assayed in quadruplicate; dissociation curves were checked and products were run in agarose gels to confirm amplification of only one product. Relative mRNA levels of β-catenin target genes (MYC, AXIN2, ASCL2, S100A6, LEF1, NOTCH2, SP5) were calculated by the 2(–∆∆Ct) method using ACTB and UBC as controls. The sequences (5′→3′) of the primers we used to measure the mRNA levels of β-catenin target and housekeeping genes are provided in Supplementary Table 5 . For statistical analysis of qRT–PCR results, we used a two-sample equal-variance t test (with a two-tailed distribution) to determine the significance of differences in gene expression.
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Anabolism
AXIN2 protein, human
beta-Catenin
Deoxyribonuclease I
Digestion
DNA, Complementary
Endoribonucleases
Gels
Gene Expression
Genes
Genes, Housekeeping
LEF1 protein, human
NOTCH2 protein, human
Oligonucleotide Primers
Reverse Transcription
RNA, Messenger
Sepharose
SYBR Green I
Technique, Dilution
trizol
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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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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TRIzol is a monophasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from various biological samples. It is a reagent designed to facilitate the disruption of cells and the subsequent isolation of RNA.
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The High-Capacity cDNA Reverse Transcription Kit is a laboratory tool used to convert RNA into complementary DNA (cDNA) molecules. It provides a reliable and efficient method for performing reverse transcription, a fundamental step in various molecular biology applications.
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Anti-LEF1 is a laboratory reagent that provides a specific detection of the LEF1 protein. LEF1 is a transcription factor involved in the Wnt signaling pathway. Anti-LEF1 can be used to study the expression and localization of LEF1 in various cellular and biological contexts.
More about "LEF1 protein, human"
Lymphoid Enhancer Binding Factor 1 (LEF1) is a crucial transcription factor that plays a pivotal role in the Wnt signaling pathway, regulating cell proliferation, differentiation, and development.
This protein is expressed in various tissues, including the lymphoid system, where it is essential for T-cell maturation and B-cell development.
LEF1 has been implicated in several disease processes, such as cancer, autoimmune disorders, and neurological conditions.
Researchers studying the LEF1 protein can leverage the PubCompare.ai tool to optimize their research workflow.
This innovative tool allows researchers to identify the best methodologies and products from literature, preprints, and patents, enhancing the reproducibility of their studies.
PubCompare.ai can help researchers compare and evaluate different experimental protocols, such as those utilizing TRIzol reagent, Lipofectamine 2000, RNeasy Mini Kit, Dual-Luciferase Reporter Assay System, Lipofectamine 3000, and TRIzol for RNA extraction and analysis.
Additionally, researchers can explore the use of various antibodies, such as Ab137872, to detect and analyze the LEF1 protein.
The incorporation of Western blotting techniques using PVDF membranes and the High-Capacity cDNA Reverse Transcription Kit for gene expression analysis can further enhance the understanding of LEF1 function and its involvement in different biological processes and disease states.
By leveraging the comprehensive insights and comparative analyses provided by PubCompare.ai, researchers can streamline their LEF1 protein research, identify the most effective methodologies, and improve the reproducibility of their studies, ultimately advancing the understanding of this crucial transcription factor and its implications in health and disease.
This protein is expressed in various tissues, including the lymphoid system, where it is essential for T-cell maturation and B-cell development.
LEF1 has been implicated in several disease processes, such as cancer, autoimmune disorders, and neurological conditions.
Researchers studying the LEF1 protein can leverage the PubCompare.ai tool to optimize their research workflow.
This innovative tool allows researchers to identify the best methodologies and products from literature, preprints, and patents, enhancing the reproducibility of their studies.
PubCompare.ai can help researchers compare and evaluate different experimental protocols, such as those utilizing TRIzol reagent, Lipofectamine 2000, RNeasy Mini Kit, Dual-Luciferase Reporter Assay System, Lipofectamine 3000, and TRIzol for RNA extraction and analysis.
Additionally, researchers can explore the use of various antibodies, such as Ab137872, to detect and analyze the LEF1 protein.
The incorporation of Western blotting techniques using PVDF membranes and the High-Capacity cDNA Reverse Transcription Kit for gene expression analysis can further enhance the understanding of LEF1 function and its involvement in different biological processes and disease states.
By leveraging the comprehensive insights and comparative analyses provided by PubCompare.ai, researchers can streamline their LEF1 protein research, identify the most effective methodologies, and improve the reproducibility of their studies, ultimately advancing the understanding of this crucial transcription factor and its implications in health and disease.