Lysates for immunoblotting were prepared from frozen cell pellets or snap-frozen xenograft tumors using RIPA buffer (Santa Cruz Biotechnology) with 1% Protease Inhibitor Cocktail (Sigma-Aldrich). Protein concentrations were determined using Bradford methodology (Bio-Rad). Proteins (50 μg per load) were resolved onto a 4–20% SDS-PAGE gradient gel (Bio-Rad), transferred onto a nitrocellulose membrane, blocked with 5% milk for 2 hrs, then incubated in primary antibody at 4 °C overnight (ZEB1 H-102, 1:200, sc-25388; ZEB2 H-260, 1:200, sc-48789; E-cadherin H-108, 1:500, sc-7870; N-cadherin, 1:500, ab18203; Vimentin V9, 1:200, sc-6260; β-Actin AC-15 1:10,000, sc-69879). All primary antibodies were obtained from Santa Cruz Biotechnology except for the anti N-cadherin antibody, which was obtained from AbCam. Membranes were then washed in TBST and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:5000, GE Healthcare) for 1 h at room temperature. Blots were developed and visualized using the Amersham™ ECL™ Western Blotting Detection Reagent kit (GE Healthcare Life Sciences). Antibody for β-actin was used as a loading control.
Anti n cadherin antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-N-cadherin antibody is a laboratory reagent used for the detection and analysis of the N-cadherin protein. N-cadherin is a cell adhesion molecule that plays a role in cell-cell interactions. This antibody can be used in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to identify and study the expression of N-cadherin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin antibody, by Abcam (8 mentions)
Anti e cadherin, by Abcam (7 mentions)
Anti vimentin antibody, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti vimentin, by Abcam (4 mentions)
Anti snail antibody, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Anti akt antibody, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by GE Healthcare (1 mentions)
Amersham ecl western blotting detection reagent kit, by GE Healthcare (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti cxcl12 antibody, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ripa buffer, by Abcam (1 mentions)
27 protocols using anti n cadherin antibody
1
Immunoblotting Assay for Epithelial-Mesenchymal Transition
2
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from MTC tissues or cells by RIPA buffer (Abcam, MA, USA) supplemented with protease inhibitor cocktail. Protein concentration was quantified by BCA assay using the standard kit (Thermo Fisher Scientific, USA). Equal amounts of protein samples were loaded and separated through SDS-PAGE. Proteins in the gels were then transferred to Nitrocellulose membranes (Millipore, MA, USA). Following that, 5 % skimmed milk was used to block the membranes for half an hour at room temperature first and then primary antibodies were added to incubate at 4˚C overnight. The membranes were then washed with TBST 3 times and then incubated with corresponding secondary antibodies (KPL, USA) at room temperature for 2 h. The membranes were washed again before visualization by the ECL kit. Primary antibodies used for the study were listed as follows: anti-Snail antibody (1: 1000, Abcam, USA); anti-N-cadherin antibody (1: 1000, Abcam, USA); anti-E-cadherin antibody (1: 1000, Abcam, USA); anti-Vimentin (1: 1000, Abcam, USA); anti-CXCL12 antibody (1: 1000, Abcam, USA); anti-GAPDH (1:2000, Abcam, USA).
3
Verifying Knockdown of Hsp70, Hdj1, and Hdj2 in C6 Cells
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The knock-down of Hsp70, Hdj1 and Hdj2 in C6 cells was verified by Western blot analysis. The C6 cells selected with puromycin (3 × 105) were lyzed on ice in solution containing 20 mM TrisHCl pH 7.5, 20 mM NaCl, 0.01% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL pepstatin. Equal amounts of protein (50 μg/lane) were electrophorezed in a 10% SDS polyacrylamide gel. Following transfer to the PVDF membrane non-specific binding was blocked with 5% fat free milk in PBS overnight at +4°C. The membranes were further incubated overnight at 4°C with monoclonal mouse antibodies as follows: J32 antibodies for Hdj1and 3C5 for Hsp70 produced at Institute of Cytology of RAS, KA2A5.6 antibodies for Hdj2 and an anti-N-cadherin antibody (both Abcam, UK).
A loading control was performed via tubulin detection (UBP-bio). Following incubation with primary antibodies, membranes were washed and incubated with horseradish peroxidase-labeled antibodies (Sigma, USA).
A loading control was performed via tubulin detection (UBP-bio). Following incubation with primary antibodies, membranes were washed and incubated with horseradish peroxidase-labeled antibodies (Sigma, USA).
4
Immunohistochemical Analysis of Cancer Protein Expression
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Immunohistochemistry (IHC) was performed to assess protein expression in cancer tissues according to previous report [26 (link)]. In brief, the formalin-fixed, paraffin-embedded tissues were cut into 4-μm sections, which were de-waxed, dehydrated, and retrieved by antigen-retrieval liquid. Then, the sections were blocked with 5% goat serum for 1 hour at room temperature, followed by incubation with indicated primary antibodies at 4°C overnight and second antibodies at 37°C for 30 min. The primary antibodies included anti-SPAG5 antibody (1:100 dilution, cat no. 14,726-1-AP, Proteintech, USA), anti-FOXM1 antibody (1:250 dilution, cat no. ab207298, Abcam, Cambridge, MA, USA), anti-ADAM17 antibody (1:100 dilution; cat no. ab39163, Abcam), ant-NOTCH1 antibody (1:150 dilution, cat no. ab52627, Abcam), anti-E-cadherin antibody (1:200 dilution; cat no. ab231303, Abcam) and anti-N-cadherin antibody (1:150 dilution; cat no. ab76011, Abcam). After that, the sections were reacted with diaminobezidin (DAB) for several seconds at room temperature and hematoxylin (Solarbio, Beijing, China) for 1 min. The staining was observed by using a microscope.
5
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Total protein was extracted from SRA01/04 cells after lysis on ice using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using a BCA kit (Beyotime Institute of Biotechnology). A total of 30 µg protein samples per well were then transferred to PDVF membranes after resolving using 10% SDS-PAGE gels. Subsequently, the membranes were washed, blocked with 5% skimmed milk for 1 h and then incubated with the following primary antibodies at 4˚C overnight: Anti-E-cadherin antibody (1:10,000; cat. no. ab40772; Abcam), anti-N-cadherin antibody (1:5,000; cat. no. ab76011; Abcam), anti-Vimentin antibody (1:1,000; cat. no. ab92547; Abcam), anti-α-smooth muscle act in (α-SMA) antibody (1:1,000; cat. no. ab265588; Abcam), anti-RhoA antibody (1:5,000; cat. no. ab187027; Abcam), anti-ROCK1 antibody (1:1,000; cat. no. ab92547; Abcam) or anti-ROCK2 antibody (1:1,000; cat. no. ab134181; Abcam). After washing with PBS, the membranes were incubated with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 2 h at room temperature. All antibodies utilized in the present study were purchased from Abcam. GAPDH was used as the loading control. The protein blots were visualized using enhanced chemiluminescence (ECL) reagent and densitometry analysis of the bands was performed using ImageJ (National Institutes of Health).
6
Western Blot Analysis of EMT Markers
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PDAC cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology). An equivalent amount of protein was loaded per lane on SDS-gel (stacking gel 4%, separation gel 10%), resolved using SDS-PAGE, transferred to PVDF membranes (EMD Millipore) and blocked using 5% skimmed milk. Subsequently, the membranes were incubated with the primary antibodies overnight at 4°C. The primary antibodies used were: Anti-HIF-1α antibody (cat. no. ab51608; 1:1,000; Abcam), anti-N-cadherin antibody (cat. no. ab202030; 1:1,000; Abcam), anti-E-cadherin antibody (cat. no. ab40772; 1:1,000; Abcam), anti-Vimentin antibody (cat. no. ab92547; 1:1,000; Abcam), anti-Snail antibody (cat. no. ab53519; 1:1,000; Abcam) or anti-β-actin antibody (cat. no. ab179467; 1:2,000; Abcam). Subsequently, the membranes were incubated with an HRP-conjugated secondary antibody (cat. no. ab205718; 1:2,000; Abcam) at room temperature for 1 h. Signals were visualized using an ECL kit (Beyotime Institute of Biotechnology). Densitometry analysis was performed using ImageJ_v1.8.0 (National Institutes of Health).
7
Protein Profiling of EMT Markers in Cell Cultures
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Total cellular protein was extracted from 2D cultures as previously described [31 (link)]. Protein concentration was determined by Lowry assay and an equal concentration of protein extracts (40 µg) was loaded on SDS-PAGE gels, transferred onto a nitrocellulose membrane and immunoblotted with primary antibodies overnight. Anti-EDB-FN antibody (G4 clone) was used at 1:1000 dilution. The following primary antibodies (1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA): anti-E-cadherin (Cat#3195), anti-Slug (Cat#9585), anti-phospho-T308-AKT (Cat#13038), anti-phospho-S473-AKT (Cat#4060), anti-pan-AKT (Cat#4691), anti-MDR1 (Cat#12683S); and anti-Histone H3 (Cat#4499) and anti-β-actin (Cat#4970) as loading controls. The anti-Phosphoepitope SR proteins (Cat#MABE50; clone 1H4) and anti-SRp40 (Cat#06-1365) antibodies were purchased from Millipore Sigma (Temecula, CA, USA) and used at 1:500 dilution. Anti-N-Cadherin antibody (Cat#76057) was purchased from Abcam (Cambridge, MA, USA) and used at 1:500 dilution. The background-adjusted pixel intensities of test proteins were normalized with those of actin controls in FIJI, and the levels were expressed as ratio of treated over non-treated cells near the respective lanes.
8
Western Blot Analysis of EMT Markers
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Proteins were isolated from cells as previously described, and were then used in the Western blot analyses (10 μg/lane) [44 (link), 45 (link)]. The membrane was probed with the following antibodies: anti-FGF2 antibody (#sc-0079, 1:1000; Santa Cruz Biotechnology, Heidelberg, CA, USA), anti-TYMS antibody (ab58287, 1:1000; Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (1:1000; Abcam), anti-N-cadherin antibody (1:1000; Abcam), anti-vimentin antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-slug antibody (1:1000; Cell Signaling Technology), anti-phosphorylated (p)Smad antibody (1:1000; Abcam), anti-twist antibody (1:1000; Abcam), anti-phospho-p44/42 MAPK (Tyr202/Tyr204) antibody (1:1000; Cell Signaling Technology), anti-p44/42 MAPK antibody (1:1000; Cell Signaling Technology), anti-phospho-Akt antibody (1:1000; Cell Signaling Technology), anti-Akt antibody (1:1000; Cell Signaling Technology), anti-MEK antibody (1:1000; Cell Signaling Technology), anti-phospho-MEK antibody (1:1000; Cell Signaling Technology), anti-phospho-EGFR antibody (Tyr1068) (1:1000; Cell Signaling Technology), and anti-EGFR antibody (1:1000; Cell Signaling Technology), and anti-β-actin (1:5000; Sigma, Saint Louis, MO, USA) was used as a loading control. Each experiment was repeated independently at least three times and one representative blot was selected for the figures.
9
Quantifying Epithelial-Mesenchymal Transition in Ovarian Cancer
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Total protein of ovarian cancer tissues, and SKOV3 and CAOV-3 cells, were extracted using radioimmunoprecipitation assay (RIPA) lysate (Beyotime, Shanghai, China). After protein quantification using the bicinchoninic acid method (Waltham, MA, USA), 80 μg protein was subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separation, and was then transferred onto polyvinylidene difluoride membranes. After that, the membranes were blocked (1 h) in TBS-T buffer containing 5% skim milk. Next, the membranes were subject to an overnight incubation (4°C) with the following primary antibodies (all from Abcam, Cambridge, MA, USA): anti-E Cadherin antibody (Cat# ab40772, 1/10000), anti-N Cadherin antibody (Cat# ab18203, 1/1000), anti-SNAIL antibody (Cat# ab53519, 1/1000), anti-Vimentin antibody (Cat# ab8978, 1/1000), and anti-3-phosphate glyceraldehyde dehydrogenase (GAPDH) antibody (Cat# ab8245, 1/500). After three Tris-Buffered Saline Tween-20 (TBST) washes, the membranes were hybridized (room temperature, 1.5 h) with the horseradish peroxidase-linked secondary antibody rabbit anti-mouse IgG H&L (Cat# ab6728, 1/2000, Abcam). Signal detection was performed using the enhanced chemiluminescence (ECL) system (Life Technologies Corporation); the relative protein levels were calculated by normalization to GAPDH.
10
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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RIPA buffer (Sigma Aldrich, Cambridge, MA) was used to lyse the cells to obtain total protein lysates. Protein concentration was measured using the BCA method (Sigma Aldrich). The quantified protein (25 μg) was transferred onto polyvinylidene fluoride (PVDF) membranes following SDS-PAGE gel electrophoresis. Then, the membrane was blocked with 5% nonfat dry milk in tri-buffered saline plus Tween (TBS-T) buffer for 2 h at room temperature and incubated with respective primary antibodies (1:1000 dilution) at 4 °C overnight, followed by Horseradish peroxidase-conjugated (HRP) secondary antibody (1:5000, Abcam, cat. no. ab7090) at room temperature for 1 h. The following primary antibodies were used: anti-HER-2 antibody (Abcam, cat. no. ab227383), anti-E-cadherin antibody (Abcam, cat. no. ab186533), anti-Snail-1 antibody (Abcam, cat. no. ab8614), anti-N-cadherin antibody (Abcam, cat. no. ab182651), anti-vimentin antibody (Abcam, cat. no. ab8805), anti-β-catenin antibody (Abcam, cat. no. ab8932), anti-GAPDH antibody (Invitrogen, cat. no. PA1–987).
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