Recombinant Human CDH2 protein, His-Fc-tagged

• Cat.No.: CDH2-315H
• Product Overview: Recombinant Human CDH2 protein(NP_001783.2) (Met 1-Ala 724) was expressed in HEK293, fused with the C-terminal polyhistidine-tagged Fc region of human IgG1 at the C-terminus.

Specification

• Species: Human
• Source: HEK293
• Tag: Fc&His
• Protein Length: 1-724 aa
• Form: Lyophilized from sterile PBS, pH 7.4.
• Molecular Mass: The secreted recombinant human CDH2 is a disulfide-linked homodimeric protein. The reduced monomer comprises 813 amino acids and has a predicted molecular mass of 89.9 kDa. As a result of glycosylation, it migrates as an apximately 114 and 119 kDa band in SDS-PAGE under reducing conditions due to glycosylation.
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method.
• Purity: > 70 % as determined by SDS-PAGE
• Storage: Store it under sterile conditions at -20°C to -80°C upon receiving. Recommend to aliquot the protein into smaller quantities for optimal storage. Avoid repeated freeze-thaw cycles.
• Reconstitution: It is recommended that sterile water be added to the vial to prepare a stock solution of 0.2 ug/ul. Centrifuge the vial at 4°C before opening to recover the entire contents.
• protein Refseq-Weblink: http://www.ncbi.nlm.nih.gov/protein/NP_001783.2
• Unit ID: P19022
• Unit ID-Weblink: http://www.uniprot.org/uniprot/P19022
• Publications:
  • A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion (2017)

Gene Information

• Gene Name: CDH2 cadherin 2, type 1, N-cadherin (neuronal) [ Homo sapiens ]
• Official Symbol: CDH2
• Synonyms: CDH2; cadherin 2, type 1, N-cadherin (neuronal); NCAD; cadherin-2; CD325; CDHN; N cadherin; N-cadherin 1; neural cadherin; neural-cadherin; cadherin 2, N-cadherin (neuronal); calcium-dependent adhesion protein, neuronal; CDw325
• Gene ID: 1000
• mRNA Refseq: NM_001792
• protein Refseq: NP_001783
• MIM: 114020

Citation

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

Journal: Nature cell biology    PubMed ID: 28218910    Data: 2018/3/1

Authors: Anna Labernadie, Takuya Kato, Xavier Trepat

Article Snippet:Bead pulling experiments were performed using magnetic tweezers as previously described 19 , 66 .Bead pulling experiments were performed using magnetic tweezers as previously described 19 , 66 .. Briefly, 4.47 μm diameter ferromagnetic beads with carboxyl surface groups (Spherotech, Lake Forest, IL) were covalently coated with a 32μg/ml solution of ，purified E-cadherin–Fc (Creative BioMart, #CDH1-274H), P-cadherin–Fc (R&D Systems, #861-PC) or N-cadherin–Fc (Creative BioMart, #CDH2-315H) proteins.. Beads were first washed with Na phosphate buffer (0.1 M, pH 8), incubated with 32 μg/ml of the Fc-tagged proteins for 5 h at 4oC and then with crosslinking buffer for 1 h (25 mM DMP, 0.2 M triethanolamine, pH 8.2).Beads were first washed with Na phosphate buffer (0.1 M, pH 8), incubated with 32 μg/ml of the Fc-tagged proteins for 5 h at 4oC and then with crosslinking buffer for 1 h (25 mM DMP, 0.2 M triethanolamine, pH 8.2).

(a) TEM image of contact (white arrows) between a CAF and a A431 cell. Image representative of 20 contacts from 3 independent experiments. Scale bar 100nm. (b) mRNA expression levels of E-,N- and P-cadherin in CAFs and A431 cells measured using QRT-PCR. Bars show average of technical triplicates. (c) Confocal immunofluorescence images of N-cadherin (red), E-cadherin (green), and CAGAP-mcherry (constitutively expressed by CAFs as a marker) in a co-culture of CAFs and A431 cells. Image representative of >4 samples. Scale bar, 5 μm. (d) Confocal immunofluorescence images of N-cadherin, P-cadherin, and CAGAP-mcherry (CAFs) in a co-culture of CAFs and A431 cells. Image representative of >4 samples. Scale bar, 5 μm. (e) SIM immunofluorescence images of N-cadherin (green), E-cadherin (yellow), β-catenin (red) and F-actin (blue) at a contact between CAF and A431 cell. Image representative of 15 samples. Scale bar is 1μm for zoomed areas, 10μm for merged overview projection. (f) STORM image of N-cadherin/E-cadherin localization at the contact between CAF and A431 cell. Image representative of 3 samples. Scale bar, 500nm. (g) Time-lapse images of a CAF expressing N-cadherin-GFP contacting A431 cells expressing E-cadherin-WT (red) (upper panels) or A431 cells expressing E-cadherin-W2A mutant (red) (lower panels), scale bars, 20μm. (h) Stacked histogram of life-time of the E-cadherin/N-cadherin junction (based on the E-Cadherin and N-cadherin fluorescent signals) at the contact between CAFs and A431 cells, for CAFs mixed with A431-EcadWT cells (rescue control, n=14 contacts from 3 independent experiments) and A431-EcadW2A mutant cells (n=28 contacts from 3 independent experiments). Data are pooled in three categories of contact life-time, from 0 to 30 min, from 30 to 60 min, and longer than 60 min duration. *** indicates p=0.0007, Chi-squared test.

(a,b) Co-cultures of CAFs from two patients with lung adenocarcinoma and H1437 cells show E-cadherin/N-cadherin junctions and β-catenin colocalization. Images representative of 2 samples for each panel. Scale bars, 5μm (see Supplementary Figure 5 for a third patient). (c) Immunostaining of the contact between cancer cells and CAFs both isolated from one patient with vulval squamous cell carcinoma. N-cadherin (red), E-cadherin (green), F-actin (blue). Images representative of 2 patient samples. Scale bar, 5μm. (d) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431 (green), CAF (red), and collagen second harmonic (magenta), arrows highlight the different tumor components. Images representative of 3 samples. Scale bar is 20μm. (e) Panels show intravital imaging xz and xy sections of a tumor growing in the mouse ear: A431-Ecad-Ruby and vulval CAF-Ncad-GFP. White arrow highlights heterotypic contact. Images representative of 3 samples. Scale bar is 20μm. (f) Images show staining of F-actin (blue), E-cadherin (green), and αSMA (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, v - vessel. Images representative of 5 samples. Scale bar, 10μm. (g) Staining of fibronectin (magenta), active integrin β1 (green), and β-catenin (red) in normal human oral mucosa and oral squamous cell carcinoma. White arrow highlights heterotypic contact between CAF and cancer cell, yellow arrow highlights integrin/ECM contact by CAF, BM – basement membrane. Images representative of 5 samples. Scale bar, 10μm.

(a) Illustration of the magnetic tweezers experimental setup. (b) Bead detachment data in A431 cells (CT), A431-EcadKO cells (EKO) and A431 cells pre-treated with E-cadherin blocking antibody (AbE). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to A431 cells after application of a force pulse. (c) Bead detachment data in CAFs transfected with siRNA Control (CT) and CAF-siNcad (siN). Percentage of beads coated with N-, E-, and P-cadherin that remained attached to CAFs after application of a force pulse. (d) Illustration of the magnetic twisting experimental setup. (e) Representative fluorescence (top) and bright field (bottom) images showing the recruitment of β-catenin, P-cadherin, and E-cadherin in A431 cells subjected to magnetic stimulation using N-cadherin-coated magnetic beads. Yellow asterisks indicate the location of the beads. Scale bars, 5μm. (f) Quantification of the recruitment of β-catenin, P-cadherin and N-cadherin mediated by N-cadherin coated beads with/without (+/- Force) mechanical stimulation. (g) Representative bead traces for A431 cells and CAFs in response to a series of force pulses applied to beads coated with N-cadherin (red), E-cadherin (blue), P-cadherin (green) or uncoated (black). Vertical bars, 200nm. (h) Stiffening of the A431 cell-bead contact defined as the time evolution of the ratio between applied force and bead displacement relative to baseline (N-,E-,P-cadherin coated beads, and uncoated beads). (i) Stiffening of the CAF-bead contact. (j) Stiffening of the cell/E-cadherin-coated bead contact for control A431 cells (A431-WT) and α-catenin mutants. (k) Stiffening of the cell/N-cadherin-coated bead contact for A431-WT cells and α-catenin mutants. (l) Stacked histogram of life-time of the E-cadherin/N-cadherin junction at the contact between CAFs and A431 cells, for CAFs mixed with A431-αcatWT cells and A431-αcatΔVBS cells. Data are pooled in three categories of contact life-time, from 0 to 30 min, from 30 to 60 min and above 60 min duration. See Supplementary Table 1 for sample numbers and statistical analysis. Error bars represent s.e.m.