For western blot experiments, 3.0×106 cells were lysed in RIPA buffer (Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The protein content of the samples was measured using BCA reagent (Thermo Fisher Scientific, Inc.). For the preparation of cell lysates to examine marker proteins, 1.0×106 cells were cultured in a 6-well plate in serum-free MEM with or without 10 ng/ml TGF-β1 for the indicated times. Cells were dissolved in SDS sample buffer containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Acrylamide gels of 12.5% (ATTO Co.) for SDS-PAGE were used for protein separation, and the proteins were subsequently transferred onto PVDF membranes (Merck). Membranes were probed with primary antibodies, including mouse anti-N-cadherin (1:250, H-2; Santa Cruz Biotechnology) and rabbit anti-Sox9 (1:1,000, AB5535; Chemicon International Inc.) antibodies, while a mouse anti-β-actin antibody (1:1,000, clone C4; Santa Cruz) was used as a loading control in siRNA experiments. The blots were then incubated with alkaline phosphatase-conjugated secondary antibody, and subsequently, signals were detected using an alkaline phosphatase substrate kit (BCIP/NBT Substrate kit; Vector Laboratories Inc.).
Mouse anti n cadherin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-N-cadherin is a primary antibody that recognizes the N-cadherin protein. N-cadherin is a cell-cell adhesion molecule involved in the formation and maintenance of cell-cell junctions. This antibody can be used to detect and study the expression and localization of N-cadherin in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti e cadherin, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Bcip nbt substrate kit, by Vector Laboratories (1 mentions)
Mouse anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl chemiluminescence system, by Cytiva (1 mentions)
Typhoon scanner, by Cytiva (1 mentions)
Mouse anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Leica tcs spe rgbv confocal microscope, by Leica Microsystems (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti smad3, by Cell Signaling Technology (1 mentions)
Sc 8426, by Santa Cruz Biotechnology (1 mentions)
Sc 8424, by Santa Cruz Biotechnology (1 mentions)
8 protocols using mouse anti n cadherin
1
Western Blot Analysis of Cell Markers
2
Protein Expression Analysis by Western Blot
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Cells or tumor tissues were homogenized and lysed with RIPA lysis buffer [100 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM sodium vanadate, and protease inhibitor]. Equal amounts of protein samples were separated by 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). The membranes were then blocked with 5% nonfat milk in Tris-buffered saline containing 0.2% Tween 20 (TBST; Invitrogen) and then incubated with specific primary antibodies at 4°C overnight. The antibodies were as follows: mouse anti-TIPE2, mouse anti-E-cadherin, mouse anti-N-cadherin, mouse anti-β-catenin, mouse cyclin D1, mouse c-myc, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using the ECL chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA). The fluorescence was scanned using a Typhoon scanner (Amersham Biosciences).
3
Immunofluorescence Analysis of Myocardial Samples
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Patient myocardial samples were formalin-fixed, paraffin-embedded, cut at a 5μm thickness and mounted on clear, plus microscope slides. Slides were deparaffinized, rehydrated, underwent antigen retrieval, then blocked at room temperature for one hour as previously described [35 (link)]. Slides were incubated overnight at 4°C with mouse anti-N-cadherin (Santa Cruz, sc-59987; 1:500) or rabbit anti-Plectin (Cell Signalling, cs-D6A11; 1:400). The following day, slides were washed and incubated with secondary antibodies (donkey anti-mouse Alexa Fluor-647 [Invitrogen, A31571; 1:500] and goat anti-rabbit Alexa Fluor-488 [Invitrogen, A11070; 1:500]), washed and cover-slipped with mounting media (Fluoroshield with DAPI, Sigma F6057). Immunoreactive signal was visualized using a Leica TCS SPE RGBV confocal microscope (Leica Microsystems) at 40X magnification. Slides were coded and analysed in a blinded fashion by three independent observers.
4
Western Blot Analysis of SMAD3, E-cadherin, and N-cadherin
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Protein was collected and extracted from A549 and H1299 cells with RIPA lysis buffer and protease inhibitor cocktail and protein phosphatase inhibitor. The samples were then transferred to PVDF membranes, following electrophoresis and the membranes were incubated with rabbit-anti SMAD3 (Cell Signaling Technology, Inc.) or mouse-anti E-cadherin or mouse-anti N-cadherin (both Santa Cruz Biotechnology, Inc.) primary antibodies(SC-8426 for E-cadherin, SC-8424 for N-cadherin) overnight at 4 °C. The following day, the membranes were incubated with indicated secondary antibodies (Santa Cruz Biotechnology, Inc, SC-2005) for 1 h at room temperature. Detection was performed using the electrochemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). β-actin (Santa Cruz Biotechnology, Inc.) was used as the internal control.
5
Whole Brain Protein Expression Analysis
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Whole brains, from mice 7-8 weeks old, were lysed in T-PER Tissue Protein Extraction Reagent (ThermoFisher) with Protease and Phosphatase Inhibitor Mini Tablets (Pierce). Tissue was homogenized with tissue grinder pestle. Cerebral cortex and cerebellum tissue was carefully dissected to ensure no contamination from ventricular structures. Samples were run by SDS-PAGE, then transferred to nitrocellulose membrane. For western blot, primary antibodies used were: BD Biosciences mouse anti-α-E-catenin (#610193), rabbit anti-α-T-catenin (polyclonal, #952), BD Biosciences mouse anti-N-cadherin (#610920), Santa Cruz Biotechnology rabbit anti-β-catenin (H-102, SC-7199), Santa Cruz Biotechnology mouse anti-δ-catenin (40.1, SC-81793), Santa Cruz Biotechnology goat anti-doublecortin (C-18, SC-8066), Cell Signaling rabbit anti-α-N-catenin (2163S), Alomone rabbit anti-GABRA2 (AGA002), and Sigma mouse anti-beta-tubulin (T4026). The secondary antibodies were: LI-COR IRDye680 Donkey anti-rabbit (926-68073) and IRDye800 Donkey anti-mouse (926-32212). Imaging performed using the Odyssey Infrared Imaging System (LI-COR).
6
Antibody Validation for EMT Markers
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Rabbit anti-Laminin C2 (SC-20776), HA-probe (SC-57592), goat anti-Snai2 (SC-10437) and mouse anti-N-Cadherin (SC-8424) were from Santa Cruz Biotechnology. Rabbit antibodies against Pam (ab86078), Fbxo45 (ab126521), GAPDH (ab37168), and mouse anti-Twist1 (ab50581) were from Abcam. Rabbit anti-E-cadherin (#3195), -Zeb1 (#3396), and mouse anti-Myc (#2276) were from Cell signaling technology. Rabbit anti-Zeb2 (NBP1-77179) was from Novus. α-tubulin (#66031-1-1g) antibody was from Proteintechnology. Anti-Flag M2 (F1804), -HA (MMS-101P) and -Vimentin V9 (V6630) mouse antibodies were from Sigma-Aldrich. Snai1 (A52437) rabbit antibodies were from ABclonal. Rabbit anti-fibronectin (GTX112794) was from GeneTex. Protein G Plus/A agarose suspension (#IP05) was purchased from Calbiochem.
7
Western Blot Analysis of EMT Markers
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Western blotting was performed as previously depicted (Dong et al., 2018 (link)). And immunoblotted with the following antibodies: anti-rabbit MT1G (1:1000, abcam, ab193329, England), anti-mouse E-cadherin (1:1000, Santa Cruz, sc-8426, USA), anti-mouse N-cadherin (1:1000, Santa Cruz, sc-8424, USA), anti-mouse snail (1:1000, Santa Cruz, sc-271977, USA), anti-mouse p-AKT (1:1000, Santa Cruz, sc-377556, USA), anti-mouse AKT (1:1000, Santa Cruz, sc-5298, USA), anti-mouse β-actin (1:1000, Santa Cruz, sc-8432, USA). Then, the PVDF membranes were washed and secondary antibodies were applied 1:5000 for 1 h at room temperature. The immunoreactions were visualized with chemiluminescent ECL reagent. Western blotting assays were performed according to a standard protocol and densitometry volume of the target bands was quantified using Fiji software.
8
Histological Characterization of ESC-Derived Teratomas
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For histological characterization, ESC-derived teratomas were fixed overnight in zinc fixative or PFA 4% at 4 • C, dehydrated in a grade alcohol series (50-100%) and embedded in paraffin. Paraffin-embedded samples were sectioned at a thickness of 4.0 μm, dewaxed, hydrated, and stained with Hematoxylin & Eosin (H&E), or May Grunwald & Giemsa solutions or processed for immunohistochemistry with anti-mouse Cytokeratin-19 (Santa Cruz Biotechnologies), anti-mouse N-cadherin (Santa Cruz Biotechnologies), anti-mouse Nestin (Santa Cruz Biotechnologies) antibodies.
To improve accessibility of antibodies to tissue antigens, tissue sections were incubated in Citrate Buffer Antigen Retrieval solution pH 6.0 at 95 • C for 20 min before immunostaining. Positive signal was revealed by 3,3 ′ -diaminobenzidine (DAB) (Roche). Sections were counterstained with Mayer's Hematoxylin before analysis by light microscopy. Images were acquired with a Zeiss Axio Imager.A2 Microscope equipped with 20 × EC Plan-NEOFLUAR and 40 × EC Plan-NEOFLUAR objectives. Image analysis was carried out using the open-source ImageJ software.
To improve accessibility of antibodies to tissue antigens, tissue sections were incubated in Citrate Buffer Antigen Retrieval solution pH 6.0 at 95 • C for 20 min before immunostaining. Positive signal was revealed by 3,3 ′ -diaminobenzidine (DAB) (Roche). Sections were counterstained with Mayer's Hematoxylin before analysis by light microscopy. Images were acquired with a Zeiss Axio Imager.A2 Microscope equipped with 20 × EC Plan-NEOFLUAR and 40 × EC Plan-NEOFLUAR objectives. Image analysis was carried out using the open-source ImageJ software.
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