33-3900

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of N-Cadherin was performed by loading 20 µg of Rat Brain (lane1), Mouse Brain (lane2) and C2C12 (lane3) lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800). Proteins were transferred to a PVDF membrane and blocked with 5 % skim milk for 1 hour at room temperature. N-Cadherin was detected at ~130 kDa using N-Cadherin Mouse Monoclonal Antibody (Product # 33-3900) at 1 µg - 3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of total Cadherin and N-Cadherin was performed by loading 2 µL SeeBlue® Plus2 Prestained Protein Ladder (Product # LC5925), 50 µg of MDCK cell lysates and 10 µg mouse heart lysate per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 1% BSA/TBST for at least 1 hour at room temperature. Total cadherin was detected using a rabbit antibody (Product # 71-7100) and N-Cadherin was detected using a mouse antibody (Product # 33-3900), both at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform. The blot was then incubated with goat anti-rabbit IgG-Alexa Fluor 790 secondary antibody (Product # A11369) and goat anti-mouse IgG-Alexa Fluor 680 secondary antibody (Product # A-21058) at a dilution of 1:10,000 for at least 1 hour. Fluorescent detection was performed using the Odyssey® CLx imaging system (Li-cor Biosciences). Image is generated by Joell Solan in Paul Lampe Lab at Fred Hutchinson Cancer Research Center.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of N-Cadherin was done on 70% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with N-Cadherin Mouse Monoclonal Antibody (Product # 33-3900) at 1 µg - 2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938)and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Product # R415, 1:300) panel c. Panel d is a merged image showing cell junctional localization of N-Cadherin. Panel e shows no primary antibody. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of N-Cadherin was done on 70% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with N-Cadherin Mouse Monoclonal Antibody (Product # 33-3900) at 1 µg - 2 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c is a merged image showing cell junctional localization of N-Cadherin. Panel d shows no primary antibody. The images were captured at 20X magnification.

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Re-morphogenesis of ARPE-19 cells treated with ITS. (A) The photomicrographs show ARPE-19 cells cultured for 4 weeks with ITS and stained for actin with Texas Red-X phalloidin. Note the tight intercellular contact, epithelial-like cellular shape, and predominantly peripheral cortical actin staining. Confocal immunofluorescence images of the same culture conditions showing beta-catenin (B) and N-cadherin (C) at the lateral membrane, CD147 (D) mostly in the apical domain and ezrin (E) in the apical domain. (F) The immunofluorescence image shows immunostaining of the alpha 1 subunit of Na + , K + -ATPase at the lateral membrane and more precisely, as observed in the XZ image, at the cell-cell contacts. (G) The beta 1 subunit is observed at the cell border and at the apical domain; the XZ image confirms both basolateral and apical staining. (H) The beta 2 subunit is observed mainly at the apical domain; the XZ image confirms apical staining. Scale bar: 10 mum. (I) Quantitative analysis of the transepithelial resistance (TER) of ARPE-19 cells during re-morphogenesis is depicted. Note a stable TER of ~80 Omega cm 2 between the second and fourth week in culture.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Rai14 is an apical and basal ES protein in the rat testis. Dual-labeled immunofluorescence analysis was used to assess co-localization of Rai14 (red) with constituent protein laminin-gamma3 chain (green), actin regulatory protein Arp3 (green), actin binding protein drebrin E (green) and actin filament cross-linking protein palladin (green) at the apical ES in stage VII tubules when these proteins were all highly expressed. It was found that Rai14 indeed partially co-localized with each of these apical ES proteins (see ""orange yellow"" in merged images) as shown in ( A ). Rai14 (red) also partially co-localized with basal ES/TJ protein ZO-1 (see ""yellow"" arrowheads), and to a lesser extent with N-cadherin (see ""white"" arrowheads) in stage IV-V tubules when these proteins were highly expressed as shown in ( B ). Bar = 10 um in ( A ) or ( B ), which applies all other images in ( A ) and ( B ).