Whole-cell lysates were separated on Tris-Glycine 4–20% gradient gels (Thermo Fisher) and transferred to nitrocellulose membranes overnight. Blots were blocked in Odyssey Blocking Buffer PBS (Licor) for 1h and incubated with primary antibodies overnight at 4 degrees, followed by 1h incubation at room temperature with secondary antibodies. Blots were imaged using the Odyssey 9210 (Licor). The control antibody was alpha-tubulin (12G10 UIOWA hybridoma bank) for all the blots except the vRPP3 blot in Fig. 1a , which we used beta-actin as the control (Sigma A1978). The other antibodies used were RAC1 (BD 610651), Cav1 (CST 3267S), AXL (CST C89E7), and E-cadherin (RD MAB1838), MEK (CST 9122), and p-MEK_S298 (CST 9128). All antibodies were used at 1:1000 dilution, except for E-cadherin (RD MAB1838), which was used at 1:250 dilution. Western blots were quantified using ImageStudioLite software. All western blots were done in triplicates, derived from same experiments, and processed in parallel. Unprocessed and uncropped blot scans can be found in the Supplementary Information (Supplementary Fig. 9 ).
E cadherin
Manufactured by R&D Systems
Sourced in United States, United Kingdom
E-cadherin is a cell-cell adhesion protein that plays a crucial role in the maintenance of cell-cell junctions and tissue integrity. It is a transmembrane glycoprotein that mediates calcium-dependent homophilic cell-cell adhesion, contributing to the formation and stabilization of adherens junctions between neighboring cells.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (9 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
N cadherin, by Abcam (3 mentions)
α smooth muscle actin, by Agilent Technologies (2 mentions)
Vimentin, by Merck Group (2 mentions)
Matrigel, by BD (2 mentions)
Tubulin, by Thermo Fisher Scientific (2 mentions)
Anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Vectastain elite kit, by Vector Laboratories (2 mentions)
P53 cm5, by Leica Biosystems (2 mentions)
Lysozyme, by Agilent Technologies (2 mentions)
Chromogranin a, by Abcam (2 mentions)
N cadherin, by Santa Cruz Biotechnology (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Vimentin, by Abcam (2 mentions)
Ifn γ, by BioLegend (2 mentions)
Fibronectin, by BD (2 mentions)
β catenin, by Santa Cruz Biotechnology (2 mentions)
59 protocols using e cadherin
1
Western Blot Analysis of Cell Signaling Proteins
2
Generating Dog BC Organoids for Drug Screening
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To generate dog BC organoids, cells from urine samples were mixed with Matrigel (BD Bioscience) and cultured with stem cell‐stimulated medium, as described previously.17 Anticancer drugs used were as follows: piroxicam; gemcitabine; vinblastine (Cayman); and cisplatin (WAKO). Antibody sources used were as follows: E‐cadherin (R&D System); CK7; Ki67 (Novus); CK20; UPK3; MMP28; TFPI2; AGPAT4 (Bioss); vimentin (Sigma‐Aldrich); α‐smooth muscle actin (SMA) (DAKO); CK5; CNN3 (GeneTex, Inc.); and CTSE (Bioworld Technology, Inc.). Fluorescent secondary antibodies used were as follows: Alexa Fluor™ 488 donkey anti‐goat IgG; Alexa Fluor 488™ goat anti‐rabbit IgG; Alexa Fluor 488™ goat anti‐mouse IgG; (Thermo Fisher Scientific Inc.); Biotinylated goat anti‐mouse IgG (Vector Laboratories, Inc.). Horseradish peroxidase ( HRP)‐conjugated anti‐rabbit IgG (Cayman); and HRP‐conjugated anti‐mouse IgG (Millipore).
3
Immunohistochemistry of Cell Markers
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Immunohistochemical (IHC) experiments were performed with 5-μm tissue sections as described [7 (link)]. Primary antibodies used include including those against E-cadherin (R&D Systems, AF648), β-catenin (Santa Cruz, sc-7199), MYC (Abcam, ab32072), EGFR (BioGenex, PU335-UP), phospho-Erk1/2 (Cell Signaling Technology, #4370) and phospho-Akt (Cell Signaling Technology, #4060). Alexa Fluor®488–, 647– or 594–conjugated secondary antibodies are from Jackson ImmunoResearch. Images were taken with a Zeiss LSM 710 confocal microscope.
4
Western Blotting Analysis of E-cadherin in C. albicans-Infected Keratinocytes
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Western blotting was performed with a standard protocol as described in (36 (link)). Protein lysate was used from VK-2 cells infected with the C. albicans end3Δ/Δ mutant or control strains for 6 h and 24 h in keratinocyte SFM media at 37°C and 5% CO2. Primary antibodies to E-cadherin (R&D systems, Minneapolis, MN) and tubulin (Invitrogen, Waltham, MA) were incubated overnight and were used at 1:500 and 1:1,000 dilutions, respectively. Anti-mouse secondary antibody (Invitrogen, Waltham, MA) was incubated for 1 h in a 1:10,000 dilution. tubulin was used as a loading control.
5
Quantifying Cell Adhesion to Extracellular Matrices
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Substrates included bovine fibronectin (Sigma), E-cadherin (R&D Systems) and BSA (Fisher). Falcon Probind 96-well plates were coated with 25- and 50-μg/ml substrates in PBS overnight at 4°C, rinsed with PBS, and blocked with 10 mg/ml BSA in PBS for 2 h at 25°C. Cells were harvested in serum free media by resuspension. 60,000 cells/well were seeded and the adhesion assay was performed for 1 h at 37°C in 5% CO2 in a humidified atmosphere. Nonadherent cells were washed away with serum free media. Adherent cells were fixed for 20 min with 4% formaldehyde in PBS, rinsed with PBS, and permeabilized with 0.1% Triton X-100 for 20 min prior to staining with Hoescht (Abcam). Excess stain was removed with PBS. Each assay point was derived from three independent experiments.
6
Histological Analysis of Mouse Intestines
7
Kidney Tissue Analysis via Immunohistochemistry
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Following bioreactor perfusion, kidneys were fixed in 10% neutral buffered formalin (NBF), imbedded in paraffin wax, and sectioned for immunochemistry (IHC). Primary antibodies for IHC were LTL (Vector labs, B1325, Newark, CA, USA), E cadherin (R&D, AF748, Santa Clara, CA, USA), Nephrin (R&D System, AF4269, Toronto, ON, Canada), KSP (Novus Biologicals, NBP1-59248, Littleton, CO, USA), AQP2 (Alomone Labs, AQP-002, Jerusalem, Israel). The primary antibodies were diluted in 0.1%FBS/0.1%Triton/PBS at a 1:100 ratio (except for E cadherin which was diluted at a 1:50 ratio). The secondary antibodies used were: Strepavidin 488 (Invitrogen, S32354, Waltham, MA, USA), Donkey anti-goat IgG Cy5 (Abcam, ab6566, Cambridge, UK), Donkey anti-sheep IgG NL557 conjugated antibody (R&D Systems, NL010), Donkey anti-rabbit IgG594 (Invitrogen, A21207), Donkey anti-rabbit IgG488 (Invitrogen, A21206). They were diluted in the same solution as the primary antibodies at a 1:500 ratio. Cell nuclei were stained with DAPI. Hematoxylin and Eosin (H&E) staining was also done to visualize tissue morphology.
Kidney was also stained with Periodic-acid Schiff (PAS) reagent staining. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was done as previously described [12 (link)].
Kidney was also stained with Periodic-acid Schiff (PAS) reagent staining. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was done as previously described [12 (link)].
8
Histological Characterization of Tumor Samples
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The spleen tumors, liver metastasis tumors, and s.c. tumors were fixated in 4% paraformaldehyde, paraffin-embedded, and sectioned at 4-μm thickness. The sections were stained with H&E or Masson Trichrome. For immunohistochemistry, antibodies against αSMA (Sigma) at 1:800, E-cadherin (R&D Systems) at 1:100, p53 (CM5) (Leica Biosystems) at 1:200, Ki67 (Abcam) at 1:1000 and Sox17 (R&D Systems) at 1:100 were used as the primary antibody. Staining signals were visualized using the Vectastain Elite Kit (Vector Laboratories). For fluorescent immunohistochemistry, Alexa Fluor 594- or Alexa Fluor 488-conjugated antibodies (Molecular Probes) were used as the secondary antibody. The numbers of p53 nuclear-accumulated cells in the liver metastasis foci were scored in 5 microscopic fields, and the ratio was calculated as the mean number of positive cells per tumor gland.
9
Comprehensive Antibody Panel for Cellular Characterization
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Antibodies against the following proteins were purchased from commercial sources: afadin, chromogranin A, and DCAMKL (Dclk) (Abcam, Cambridge, UK); E-cadherin (R&D Systems, Minneapolis, MN, USA and BD Biosciences, San Jose, CA, USA); ZO-1 (Sanko-junyaku, Tokyo, Japan); Ki-67 (Novocastra Laboratories, Newcastle Upon Tyne, UK); lysozyme (DAKO, Glostrup, Denmark); cleaved caspase3 (Cell Signaling, Beverly, MA, USA); Rap1 (Millipore Corporation, Billerica, MA, USA); EphB3 (Abcam and R&D Systems); and EphB2 and ephrinB1 (R&D Systems). Alexa Fluor and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Millipore Corporation and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.
10
Tryptic Digestion of E-cadherin and HtrA
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Tryptic digestion was performed on three different samples (sample volume: 200 μl). Sample 1 contained human recombinant E-cadherin Fc His6 (22.5 ng/μl E-cadherin D155–I707, R&D Systems) in 50 mM HEPES buffer (HEPES buffer 1 M solution, pH 7.3, Fisher Scientific), 0.5 mM MgCl2 (Sigma-Aldrich Chemie GmbH) and 1 mM CaCl2 (Sigma-Aldrich Chemie GmbH, pH 7.3). Sample 2 contained HtrA (2.5 ng/μl in 50 mM HEPES buffer, pH 7.3). Sample 3 included HtrA and human recombinant E-cadherin Fc His6 at a ratio of 1:10 (2.5:22.5 ng/μl in 50 mM HEPES buffer, pH 7.3). All samples were incubated for 12 h at 37 °C in a horizontal shaker. Samples 1 and 2 were applied in sextuplicate, and sample 3 was done in triplicate. Finally, 2 μl of sequencing-grade modified porcine trypsin was added (stock solution of 50 ng/μl in 50 mM HEPES, 1 mM CaCl2, pH 7.3, Promega). Protease digestion was carried out overnight at 37 °C in a horizontal shaker. After tryptic digestion, Samples 2 and 1 were mixed at a ratio of 1:10 to obtain Sample 4. Peptides were desalted, purified and concentrated using C18 microcolumns OMIX tips (Agilent Technologies). After lyophilisation, peptides were stored at –20 °C.
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