Whole cultured cells were homogenized in 0.1% SDS and 1 mM PMSF (phenylmethylsulfonyl fluoride) and centrifuged at 12, 000 g for 10 min. Protein extracts were subjected to SDS-PAGE and analyzed using the following primary antibodies: LEF1 antibody (Abcam, ab137872), YAP1 antibody (Abcam, ab52771), TAZ antibody (Proteintech, 23306-1-AP), E-cadherin antibody(Abcam, ab40772), N-cadherin antibody (Abcam, ab18203) and GADPH antibody (Abcam, ab8245). Then, the membranes were incubated with secondary antibodies (CST,7076,7074) at room temperature for 1 hour. All experiments were performed in triplicate.
E cadherin antibody
Manufactured by Abcam
Sourced in United Kingdom, United States, China
E-cadherin antibody is a protein used to detect and quantify the expression of the E-cadherin protein in biological samples. E-cadherin is a cell-cell adhesion molecule that plays a critical role in the maintenance of cell-cell junctions and epithelial cell polarity.
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Lab products found in correlation
N cadherin antibody, by Abcam (5 mentions)
Vimentin antibody, by Abcam (3 mentions)
β actin antibody, by Abcam (3 mentions)
N cadherin, by Abcam (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lef 1 antibody, by Abcam (1 mentions)
Yap1 antibody, by Abcam (1 mentions)
Gadph antibody, by Abcam (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Egr1 antibody, by Abcam (1 mentions)
Sox10 antibody, by Abcam (1 mentions)
Tgf β1 antibody, by Abcam (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Las x software, by Leica Microsystems (1 mentions)
Twist antibody, by Abcam (1 mentions)
Gsk3β antibody, by Abcam (1 mentions)
Bax antibody, by Abcam (1 mentions)
20 protocols using e cadherin antibody
1
Protein Expression Analysis of Cultured Cells
2
Protein expression analysis in biological samples
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Cells were collected and lysed on ice in RIPA buffer containing a protease inhibitor cocktail. Protein concentration was assessed and 100 µg of protein from each sample was loaded and separated by 10% polyacrylamide gel electrophoresis and transferred onto PVDF membranes. After incubation with the specific primary antibodies: REC8 antibody (1:1,000; rabbit monoclonal, cat. no. ab192241; Abcam, San Francisco, CA, USA), EGR1 antibody (1:1,000; rabbit polyclonal, cat. no. ab6054; Abcam), TGFβ1 antibody (1:1,000; rabbit polyclonal, cat. no. ab92486; Abcam), SOX10 antibody (1:1,000; rabbit polyclonal, cat. no. ab108408; Abcam), vimentin antibody (1:1,000; rabbit monoclonal, cat. no. ab92547; Abcam), E-cadherin antibody (1:1,000; rabbit polyclonal, cat. no. 3195; Cell Signaling Technology, Danvers, MA, USA), GAPDH antibody (1:4,000; rabbit polyclonal, cat. no. 2118; Cell Signaling Technology), ATG12 antibody (1:1,000; mouse monoclonal, cat. no. sc-271688; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000; cat no. A4416 and A6154; Sigma-Aldrich). ECL luminescent reagent (cat. no. 32109; Invitrogen/Thermo Fisher Scientific) was added and the signals were recorded on X-ray film in a darkroom. GAPDH was used as the loading control.
3
Multicolor Immunofluorescence Staining Protocol
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Antibodies were purchased from Biolegend. Twenty-five× 104 cells were first seeded on cover glasses in 6-well plates. After 48 h, the culture medium was aspirated, and cells were fixed with 2% paraformaldehyde for 20 min at room temperature. After washing twice by PBS, the cells were incubated in 0.5% Triton X-100 in PBS for 10 min for permeabilization and blocked by cell staining buffer (Biolegend, San Diego, USA) for 30 min. Then the coverslips were transferred into a humidified chamber and incubated with Alexa Fluor 488-conjugated anti-human Ki-67 antibody, Alexa Fluor 594 anti-human Epithelial cadherin (E-cadherin) antibody, Alexa Fluor 647 anti-GATA3 antibody and Alexa Fluor 488 anti-Vimentin antibody overnight at 4 °C, or with Alexa Fluor 488-conjugated Flash Phalloidin (F-Actin) in room temperature for 1 h. After washing twice, the coverslips were mounted on glass slides with 25 μl of mounting medium with 4′,6-diamidino-2-phenylindole (Abcam, Cambridge, UK). The slides were analyzed by a Leica SP8 confocal microscope. Images were taken using LAS X software (Leica Microsystems, Wetzlar, Germany).
4
Evaluating EMT and Apoptosis Markers
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The cells of each group were collected and lysed, while the total protein was extracted. The protein concentration was examined by BCA, and we adjusted the protein concentration of each group. We loaded samples 30 μg/well, and electrophoresis was performed with SDS-PAGE gel. The protein was transferred to the PVDF membrane, sealed with skim milk at 25°C, incubated with primary antibody at 4°C overnight, incubated with HRP-linked secondary antibody, developed and photographed with an ECL kit. Primary antibodies included Snail antibody (ab216347), Twist antibody (ab50887), E-cadherin antibody (ab231303), N-cadherin antibody (ab76011), GSK3β antibody (ab32391), β-catenin antibody (ab32572), Bax antibody (ab32503), Bcl-2 antibody (ab182858), and GAPDH antibody (ab8245), all derived from Abcam.
5
GATA3 Regulation of Epithelial-Mesenchymal Transition
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U2OS and Saos2 cells were transfected with FLAG-GATA3 plasmid or GATA3 siRNA for 48 hours. Subsequently, cells were collected and lysis by RIPA protein extraction reagent supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The concentration of protein was quantified using the BCA Protein Quantifcation kit (Pierce, Waltham, MA, USA) according to the manufacturer’s instructions. A total of 40 µg protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Sigma-Aldrich Co., St Louis, MO, USA). The membranes were blocked with 5% skim milk (OriGene Technologies, Inc., Beijing, People’s Republic of China) for 1 hour at room temperature. Subsequently, the membranes were incubated with antibodies, such as GATA3 (1:2,000, Abcam, Cambridge, UK), E-cadherin antibody (1:2,000, Abcam), N-cadherin antibody (1:2,000, Abcam), vimentin antibody (1:2,000, Abcam), slug (1:1,000, Abcam), snail (1:2,000, Abcam), twist1 (1:1,000, Abcam), and β-actin antibody (1:5,000, Abcam) overnight at 4°C. After washing with TBST buffer three times, the membranes were incubated with horseradish peroxidase-coupled (HRP) secondary antibody (1:5,000, Abcam) at room temperature for 1 hour. Finally, the blots were visualized using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer, Inc., Waltham, MA, USA).
6
Immunohistochemical Staining of ZEB-1 and E-cadherin
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The streptavidin–biotin–peroxidase immunohistochemical staining method was used in this study. Four-micrometer sections of formalin-fixed paraffin-embedded tissue were mounted on poly-L-lysine-coated slides. The slides were deparaffinized, and then endogenous peroxidase activity was blocked with 3% hydrogen peroxide in 50% methanol for 10 min at room temperature. The slides were rehydrated and washed with phosphate-buffered saline (PBS) and then pretreated with citrate buffer (0.01 M citric acid, pH 6.0) for 20 min at 100°C in a microwave oven. After nonspecific binding sites were blocked by incubating in 2% normal goat serum in PBS for 15 min at 37°C, the slides were incubated overnight at 4°C with the primary rabbit ZEB-1 antibody (dilution 1:100; Abcam) and rabbit E-cadherin antibody (dilution 1:200; Abcam). The slides were washed with PBS and incubated with biotin-labeled secondary antibody (dilution 1:100; Zhongshan Biotechnology) for 1 hour at 37°C and then incubated with avidin–biotin-conjugated peroxidase and diaminobenzidine (Zhongshan Biotechnology), and counterstained with hematoxylin. Slides were dehydrated with different concentrations of alcohol and soaked in xylene, and then mounted with neutral balsam and visualized using light microscope.
7
Immunofluorescence Staining of E-cadherin
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Cells were incubated in a culture dish with a cover glass, and the culture medium was removed when the cover glass was full of cells. Then, cells were washed with PBS, fixed with 4% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h. The cells were then incubated overnight at 4℃ with E-cadherin antibody (1:100, ab194982, Abcam). Subsequently, cells were observed under a fluorescence microscope (Olympus CX23, Tokyo, Japan). Image was taken with an Operetta High Content Analysis System (Perkin Elmer Foster City, CA, USA).
8
Immunohistochemical Analysis of Kidney Tissue
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The 4-μm formalin-fixed paraffin-embedded renal tissue sections were dewaxed and placed into water. After antigen repairing, the tissue sections were blocked with 5% bovine serum albumin and then incubated with HBsAg and E-cadherin antibody (Abcam, Cambridge, MA, UK) overnight at 4 °C. After three times washes with phosphate-buffered saline, secondary antibody was added and incubated for 2 h at room temperature (RT), and then stained with 4′,6-diamidino-2-phenylindole (Invitrogen) for 2 min. Images were captured using a fluorescence microscope (Olympus BX51TF, Tokyo, Japan).
9
Immunohistochemical Analysis of Cell Adhesion Proteins
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After sacrifice, control and plasma tumor sample tissues were fixed in formalin for the preparation of paraffin sections. Paraffin-embedded tissue sections were deparaffinized in xylene, 95, 90 and 70% ethanol, followed by PBS. Tissue sections were immunostained overnight at 4 °C with the E-cadherin antibody (1:400; abcam, Cambridge, UK), N-cadherin (1:400; abcam) antibody. After PBS washing, 1:200 dilution of biotinylated goat anti-mouse IgG antibody in blocking solution was applied to the sections and incubated for 30–40 min. Following with PBS, ABC reagent was applied to the sections and incubated further for 30 min. Color reaction was performed with 3, 30-diaminobenzidine (vector laboratories, Seoul, Korea) and slides were washed twice with PBS. After counter-staining with hematoxylin and clearing with graded ethanol series and xylene, sections were mounted with Canada balsam. Images were photographed using IX71 microscope (Olympus, Tokyo, Japan) equipped with DP71 digital imaging system (Olympus). For immunohistochemistry, E-cadherin (ab15148), N-cadherin (ab18203), Vimentin (ab137321), CD86 antibody (NBP2-25208-novus biologicals, Centennial, CO, USA) were used.
10
Pluripotent Stem Cell Characterization
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We obtained Roswell Park Memorial Institute (RPMI) 1640 medium, TRizol total RNA extraction reagent, and diethyl pyrocarbonate (DEPC) water (TIANGEN BIOTECH, Beijing, China); Trans2K Plus II DNA Marker and FBS (Qiagen, Dusseldorf, Germany); goat anti-mouse secondary antibody, Oct4 antibody, Sox2 antibody, CD44 antibody, E-cadherin antibody, and β-actin antibody (Abcam, Cambridge, USA); PrimeScript RT Enzyme Mix I (x 200) and SYBR Green 820A (Takara, Tokyo, Japan); and Oct4 primer, Sox2 primer, CD44 primer, E-cadherin primer, KLF4 primer, c-Myc primer, B27 additive, human epidermal growth factor (EGF), and human basic fibroblast growth factor (bFGF; Invitrogen, Carlsbad, USA).
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