Human colon cancer cell line SW620 was purchased from the American Type Culture Collection (ATCC), and HCT116 p53−/− cells were generous gifts from Dr. Bert Vogelstein at the John Hopkins Medical Institutes. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen), at 37 °C, 5% CO2. Human recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN). The antibodies were purchased from Santa Cruz (anti-E-cadherin, and anti-Vimentin), Sigma-Aldrich (anti-PHLDB2), Thermo Fisher (anti-MDM2) and Millipore (anti-glyceraldehyde-3-phosphate dehydrogenase, GAPDH). PHLDB2 siRNAs and non-specific control oligo were purchased from Ambion. Transfection was performed using Lipofectamine 2000 (Invitrogen) per manufacture’s instruction.
Anti e cadherin
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Anti-E-cadherin is a lab equipment product used to detect and measure the levels of E-cadherin, a cell-cell adhesion protein, in biological samples. It functions as a tool for researchers to investigate cellular processes and pathways involving E-cadherin expression.
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Lab products found in correlation
Anti β actin, by Merck Group (9 mentions)
Anti vimentin, by Merck Group (6 mentions)
Anti zo 1, by Thermo Fisher Scientific (4 mentions)
Anti β catenin, by Merck Group (3 mentions)
Anti n cadherin, by Merck Group (3 mentions)
Anti snail, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Anti vimentin, by Cell Signaling Technology (3 mentions)
Tgf β1, by R&D Systems (2 mentions)
Anti snail, by Cell Signaling Technology (2 mentions)
Anti occludin, by Thermo Fisher Scientific (2 mentions)
Anti hsp70, by Cell Signaling Technology (2 mentions)
Anti slug, by Merck Group (2 mentions)
Anti gm130, by Merck Group (2 mentions)
Anti alix, by Santa Cruz Biotechnology (2 mentions)
Anti zeb2, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Anti zo 1, by Merck Group (2 mentions)
Anti n cadherin, by Cell Signaling Technology (2 mentions)
34 protocols using anti e cadherin
1
Culturing Human Colon Cancer Cell Lines
2
Western Blot Analysis of Cell Signaling Proteins
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The cells were pyrolysis in RIPA buffer containing protease inhibitors. The lysates were centrifuged at 10,000 rpm for 10 min at 4°C. After centrifuge, the supernatant were collected. Total of 50 µg protein was uploaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a PVDF membrane. The membrane was blocked using 5% fat-free milk at room temperature for 2 h. The concentration of antibody for incubated is as following: anti-E-cadherin (1:200), anti-Cyclin D1 (1:1,000), anti-C-Myc (1:1,000), anti-β-catenin (1:500), anti-β-actin (1:1,000) (Sigma, USA) at 4°C for overnight. After three washes for 5-min in TBST, the membrane was incubated in peroxidase-conjugated goat anti-mouse/rabbit IgG or peroxidase-conjugated rabbit anti-goat IgG (Abcam, US) for 2 h at room temperature. An electrogenerated chemiluminescence system was used to visualize the proteins.
3
Leukemia Cell Adhesion Assay
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The leukemia cells treated with and without 1 μg/mL PMA were cultured in flasks in advance for 48 hours. The cells were trypsinized for 2 minutes, centrifuged, and resuspended in fresh Gibco RPMI 1640 medium with 10% FBS. The cells were then counted with a hemocytometer to a concentration of 1×106 cells/mL, and 100 μL of the culture medium containing the cells was added to each well of the 96-well microplates, which were precoated with 4 μg/mL BSA proteins, fibronectin, collagen I, and Anti-E-cadherin (Sigma-Aldrich, St Louis, MO, USA). The cells were incubated for 30 minutes for cell attachment, and the remaining suspended cells were removed. The attached cells were measured using the MTT assay at 570 nm wavelength absorbance in an Asys UVM 340 microplate reader (Biochrom, Cambridge, UK).
4
Immunofluorescence Analysis of EMT Markers
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The cells were placed on coverslips and exposed to TGF-β1 (5 ng/mL) after transfection with miR-29b mimic, miR-29b inhibitor, or siHSP47. Subsequently, The cells were permeated with 0.2% Triton X-100 in 1% bovine serum albumin for 10 min, blocked by 5% bovine serum albumin for 1 h at room temperature, incubated overnight at 4 °C with monoclonal anti-HSP47, anti-E-cadherin, and anti-vimentin antibodies (Sigma-Aldrich), then fixed with 4% paraformaldehyde. Goat anti-mouse Alexa 488 (Invitrogen) and goat anti-rabbit Alexa 555 (Invitrogen) secondary antibodies were added to the cells for incubation. Finally, the nucleus was counterstained with 4′,6-diamidino-2-phenylindole (Invitrogen) and the stained cells were visualized using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).
5
Protein Expression Analysis in Cell Lysates
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After the cells were collected, total proteins were extracted using cell lysis, and the DC Protein Assay kit was then used to determine the protein concentrations. A total of 50 µg/well protein was then used for gel electrophoresis on 10% SDS-PAGE gels, and the proteins isolated after electrophoresis were transferred onto polyvinylidene difluoride film (PVDF; Millipore, Billerica, MA, USA). Blocking buffer was then added for 1 h at room temperature, followed by the addition of the primary antibodies (anti-ANXA1, Cat. no. SAB1405457, 1:1,000; anti-Snail, WH0006591M5, 1:2,000; anti-E-cadherin, WH0000999M1, 1:2,000; and the internal control β-actin, A1978, 1:3,000) (all from Sigma-Aldrich, St. Louis, MO, USA) for overnight culture at 4°C with mild shaking. The membranes were then washed and 2% BSA-containing horseradish peroxidase-labeled secondary antibody (goat anti-mouse, Cat. no. SAB4600316, Sigma-Aldrich) was then added for 1 h at room temperature. The membranes were then washed again, and then exposed and developed. Quantity One image analysis software was then used for analysis, and each experiment was repeated 3 times.
6
Protein Expression Analysis Protocol
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Cells were dissociated in cracking 50 mM Tris-HCl (pH 7.4) buffer containing 2-mercaptoethanol (100 mM), SDS (2% w/v), and glycerol (10%). After separation using SDS-PAGE, proteins were electrotransferred onto PVDF membranes (Millipore, USA). The membranes were sealed in milk and incubated with primary antibodies, including anti-type I collagen, anti-E-cadherin, anti-Desmin, anti-Smad7, anti-DNMT1, and anti-β-actin (Sigma, St Louis, MO, USA) antibodies, at 4°C overnight. Subsequently, they were incubated with the secondary antibody, goat anti-rabbit IgG (1:2000, Rockland), for almost 1 h at 37°C. β-actin protein levels were used as the internal control.
7
Immunofluorescence Staining in Mouse Embryos
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Immunofluorescence was performed as previously described (Dietrich and Hiiragi, 2007 (link)). The following antibodies and dilutions were used: monoclonal mouse anti-CDX2 (MU392-UC, BioGenex) 1:200, rabbit monoclonal anti-CDX2 (ab76541, Abcam) 1:200, mouse monoclonal anti-YAP (sc-101199, Santa Cruz Biotechnology) 1:200, rabbit polyclonal anti-pERM (3141, Cell Signalling) 1:250, rat monoclonal anti-E-Cadherin (U3254, Sigma) 1:250, mouse monoclonal anti-TEAD4 (ab58310, Abcam) 1:100, rabbit polyclonal anti-DsRed (632496 living colors Clontech) 1:400, goat polyclonal anti-GFP (R1091P, Acris, Origene) 1:200, rat monoclonal anti-HA (11867423001, Sigma) 1:200. Secondary Alexa Fluor conjugated antibodies (Life Technologies) were used at 1:1000. Nuclei were visualized by incubating embryos in DAPI at 1 μg/ml.
8
Protein Expression Analysis via Western Blot
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Western blot analyses were performed according to standard protocols as previously described [11 (link)].
Anti-E-cadherin, Anti-N-cadherin, Anti-vimentin and Anti-PHF14 were purchased from Sigma.
Anti-E-cadherin, Anti-N-cadherin, Anti-vimentin and Anti-PHF14 were purchased from Sigma.
9
Immunofluorescence Analysis of E-cadherin in A549 Cells
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For immunofluorescence analysis, A549 grown on 8-well chamber slides were fixed for 20 min in cold methanol at −20 °C and rinsed five times in PBS. Afterwards, the cells were permeabilized for 5 min with 0.5% TritonX-100 in PBS at RT, washed twice with PBS and incubated overnight at 4 °C with anti-E-cadherin (1:500; U3254, Sigma). Cells were then washed three times with PBS and incubated with anti-rat conjugated with Alexa Fluor®Dye 555 (Thermo Scientific) for 1 h at RT in the dark. Cells were washed three times in PBS and mounted with fluorescence vectashield mounting medium with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for the counterstaining (Vector). For microscopic inspection, an Olympus fluorescence microscope was used.
10
Immunostaining of Mouse Mammary Epithelial Cells
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Mouse MECs were immunostained as previously described1 . Briefly, MECs were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and then, stained with the following antibodies: anti-E-cadherin (1:50; Sigma) and anti-ZO-1 (5 µg/mL; Life Technologies). Primary antibodies were visualized using secondary antibodies conjugated with Alexa Fluor® 488 or Alexa Fluor® 568 (Life Technologies) and counterstained with DAPI nuclear stain (1 µg/mL; Molecular Probes). Cells were examined using a Leica Inverted Confocal Microscope SP8 (Leica Microsystems, Wetzlar, Germany).
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