Human/Mouse/Rat N-Cadherin Antibody

Detection of Human, Mouse, and Rat N‑Cadherin by Western Blot. Western blot shows lysates of A549 human lung carcinoma cell line, HeLa human cervical epithelial carcinoma cell line, C2C12 mouse myoblast cell line, and rat brain tissue. PVDF Membrane was probed with 0.5 µg/mL of Sheep Anti-Human/Mouse/Rat N-Cadherin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6426) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for N-Cadherin at approximately 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

N‑Cadherin in A549 Human Cell Line. N-Cadherin was detected in immersion fixed A549 human lung carcinoma cell line using Sheep Anti-Human/Mouse/Rat N-Cadherin Affinity-purified Polyclonal Antibody (Catalog # AF6426) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to cell surfaces. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

N‑Cadherin in Human Heart N-Cadherin was detected in immersion fixed paraffin-embedded sections of human heart using Sheep Anti-Human/Mouse/Rat N-Cadherin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6426) at 1 µg/mL overnight at 4 °C. Tissue was stained using the Sheep IgG VisUCyte HRP Polymer Antibody (Catalog # VC006). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to intercalated disks between the myocardial cells. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Human N‑Cadherin ELISA Standard Curve. Recombinant Human N-Cadherin protein was serially diluted 2-fold and captured by Mouse Anti-Human N-Cadherin Monoclonal Antibody (Catalog # MAB13883) coated on a Clear Polystyrene Microplate (Catalog # DY990). Sheep Anti-Human/Mouse/Rat N-Cadherin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6426) was biotinylated and incubated with the protein captured on the plate. Detection of the standard curve was achieved by incubating Streptavidin-HRP (Catalog # DY998) followed by Substrate Solution (Catalog # DY999) and stopping the enzymatic reaction with Stop Solution (Catalog # DY994).

Detection of N‑Cadherin in A172 cells by Flow Cytometry. A172 cells were stained with Sheep Anti-Human/Mouse/Rat N‑Cadherin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6426, filled histogram) or isotype control antibody (Catalog # 5-001-A, open histogram), followed by Fluorescein-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # F0125). View our protocol for Staining Membrane-associated Proteins.

Detection of Human N-Cadherin by Western Blot N-Cadherin Depletion Rescues the Migration Defect in Rab14- and FAM116-Depleted Cells(A and B) A549 cells were treated with control, Rab14, FAM116A, N-cadherin, E-cadherin, and FGFR2 siRNA duplexes alone or in the combinations indicated for 72 hr (A). The cells were then western blotted for N-cadherin, E-cadherin, or tubulin as a loading control or (B) fixed and then stained with DAPI and antibodies to N-cadherin. Scale bar indicates 10 μm.(C) A549 cells were treated with control, Rab14, FAM116A, N-cadherin, E-cadherin, and FGFR2 siRNA duplexes alone or in the combinations indicated for 72 hr. The cell monolayers were then scratched and imaged for 16 hr. Cell migration was measured and is plotted on the graph with error bars to show the standard error of the mean (n = 3).(D) Images are shown from the 0 and 16 hr time points for the Rab14- and FAM116A-depleted cells in the presence and absence of N-cadherin siRNA. Scale bar indicates 50 μm.See also Figure S5. Image collected and cropped by CiteAb from the following publication (https://linkinghub.elsevier.com/retrieve/pii/S1534580712001876), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human N-Cadherin by Western Blot Abnormal Adherens Junctions in Rab14- and FAM116-Depleted Cells(A) A549 cells were treated with control, Rab4, Rab11, Rab14, FAM116A, and Avl9 siRNA duplexes for 72 hr. The cells were lysed in SDS-PAGE sample buffer and then western blotted as indicated in the figure. Tubulin was used as a loading control.(B) A549 cells were treated with control, Rab14, and FAM116A siRNA duplexes for 72 hr, fixed, and then stained with DAPI and antibodies to N-cadherin.(C) A549 cells were treated with control, Rab4, Rab11, Rab14, FAM116A, and Avl9 siRNA duplexes for 72 hr. The cell monolayers were then scratched, samples fixed at 0, 4, 8, and 16 hr, and then stained with DAPI and antibodies to N-cadherin. Images are oriented so the wound is to the right. Scale bar indicates 10 μm in all images.See also Figure S6. Image collected and cropped by CiteAb from the following publication (https://linkinghub.elsevier.com/retrieve/pii/S1534580712001876), licensed under a CC-BY license. Not internally tested by R&D Systems.