Histological analysis of primary tumors was performed for a randomly selected subset of CTRL and miR-127PD tumors. Immunohistochemistry was performed as described (21 (link)). Anti-Ki-67 (1:500) (Cell Signaling Technologies, Cat# 9027), anti-cleaved Caspase 3 [Asp175] (1:500) (Cell Signaling Technologies, Cat# 9661), anti-human mitochondria [113-1] (1:1000) (Abcam, Cat# ab92824), anti-CyclinD1 (Novus Biologicals, Cat# NB100-79920) (1:100), anti-Vimentin (Gentex, Cat# GTX100619) (1:300), anti-Twist1/2 (Genetex, Cat# GTX127310) (1:200), and anti-N-Cadherin (Epitomics, Cat# 2447-1) (1:100), primary antibodies were used. An internal negative control (no primary antibody) was included with each trial. Images of 4 – 6 randomized fields of view per animal were captured then quantified using H DAB color deconvolution software in Image J Fiji (National Institutes of Health, RRID: SCR_002285). For analysis of tumor cell infiltration into lymph node tissue (n = 14), Image J software was used to quantify the total area of the lymph node compared to the total area comprised of tumor cells.
Anti n cadherin
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Anti-N-cadherin is a laboratory reagent used for the detection and quantification of N-cadherin protein. N-cadherin is a cell adhesion molecule involved in cell-cell interactions. This antibody can be used in various immunoassay techniques to study the expression and localization of N-cadherin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin, by Abcam (153 mentions)
Anti vimentin, by Abcam (107 mentions)
Anti gapdh, by Abcam (59 mentions)
Pvdf membrane, by Merck Group (35 mentions)
Ripa lysis buffer, by Beyotime (27 mentions)
Anti β actin, by Abcam (25 mentions)
Anti e cadherin, by Cell Signaling Technology (20 mentions)
Anti β catenin, by Abcam (17 mentions)
Anti snail, by Abcam (16 mentions)
Anti slug, by Abcam (15 mentions)
Anti bcl 2, by Abcam (14 mentions)
Anti cyclin d1, by Abcam (13 mentions)
Anti zeb1, by Abcam (12 mentions)
Anti mmp9, by Abcam (12 mentions)
Anti bax, by Abcam (12 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Anti vimentin, by Cell Signaling Technology (11 mentions)
Anti ki67, by Abcam (10 mentions)
Anti bax, by Cell Signaling Technology (10 mentions)
Anti mmp2, by Abcam (10 mentions)
245 protocols using anti n cadherin
1
Histological Analysis of Tumor Samples
2
Western Blot Analysis of EMT Markers
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SW480 and HCT116 cells were seeded in 6-well plates until they grew with adherence, the various conditioned media was replaced as described previously and incubated for 24 hours, and cell extracts were prepared in ice-cold lysis buffer containing protease inhibitor. The cell proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride (PVEF) membranes (Millipore). Membranes were further incubated sequentially with specific antibodies including anti-E-cadherin (1:1000, Pro780, Cell Signaling Technology), anti-N-cadherin (1:1000, EPR1791–4, Epitomics), anti-Vimentin (1:1000, EPR3776, Epitomics), anti-MIF (1:1000, Santa Cruz Biotechnology), anti-p-Cofilin (1:1000, 77G2, Cell Signaling Technology), and anti-F-actin (5 μg/ml, 4E3.adl, Abcam). After primary antibodies were incubated, the blots were subsequently incubated with appropriate secondary antibodies. Protein bands were visualized with ECL reagent (Thermo Scientific Inc.) and a Bio-Rad image acquisition system (Bio-Rad Laboratories). The protein bands was quantified using densitometric scanning software, and relative protein abundance was determined by normalization with tubulin or GAPDH.
3
Quantitative Western Blot Analysis
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A total of 50 mg protein was separated by denaturing 8–15% SDS–polyacrylamide gel electrophoresis. Western analysis was conducted as previously described [12] (link). The films were analyzed by densitometry with a VersaDoc Imaging System; Model 3000 (BioRad) using Quantity One software. Analysis of variance with Bonferroni correction for multiple tests was used to determine significance. The primary antibodies used were anti-Ncadherin (1∶5000), anti-Ecadherin (1∶2000), anti-fibronectin (1∶1000), anti-vimentin (1∶1000) and anti-actin (1∶2000) as an endogenous control, all from Epitomics Biotechnology (Epitomics).
4
Molecular Profiling of Epithelial-Mesenchymal Transition
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Experimental cancer cells (after culturing in HUVEC-CM for 21 days) and respective control cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). Western-blot analysis was performed by established protocols 12 . Anti-α-catenin, anti-E-cadherin, anti-ZEB-1, ZEB-2, Snail, Slug, anti-ZO-1 and anti-Laminin A1 primary antibodies were purchased from (Abcam); anti-MMP-1, -2, -3, -11, -12, -13, -17 and -21, anti- ITGA6, B1, B3, B4, B7, anti-FAK, P-FAK-Y397, anti-Src, P-Src-Y418, P-Src-Y529, anti-fibronectin, anti-Laminin B3, anti-Wnt-2, anti-Wnt-5B, anti-Wnt-16, anti-TGF-β, anti-β-catenin and anti-N-cadherin primary antibodies were purchased from (Epitomics); anti-Cdc-2, P-Cdc-2-Tyr15, CDK4, CDK6, Cyclin A, Cyclin D1, Cyclin D3, Cyclin E2, P15, P16, P21, P27, P53, Rb, P-Rb-S811, P-Rb-S780; anti-Bcl-xl, Bad, P-Bad-Ser112, Bak, Mcl-1, Puma and anti-β-actin were from (Cell Signaling Technology).
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted with sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol, and 5% 2-β-mercaptoethanol), and its concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo, Massachusetts, MA). Total protein was subsequently separated on 6%-12% SDS–polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk and incubated with primary antibodies, followed by incubation with anti-mouse or rabbit IgG secondary antibodies. Bands were detected by enhanced chemiluminescence, and GAPDH or α-tubulin served as the loading control. Anti-ZNF488 was purchased from Abcam, Cambridge, UK; anti-ZO1, anti–E-cadherin, and anti–α-catenin from BD Biosciences, Franklin Lakes, NJ; anti–α-tubulin, anti-Slug, anti-Snail, anti–phospho-GSK3β Ser9, and anti-GSK3β from Cell Signaling Technology, Boston, MA. Anti-vimentin (Novagen, South Africa), anti–N-cadherin (Epitomics, Burlingame, CA) and anti–β-catenin (Santa Cruz, Shanghai, China) were purchased.
6
Immunohistochemical Staining of Cell-Cell Adhesion Markers
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Sections (4-μm) of the tissue microarray block were cut and placed on glass slides for IHC staining. A mouse monoclonal anti-E-cadherin antibody (Maxim, 1:100 dilution) was used for E-cadherin staining with an Elivision plus IHC kit (Maxim, Fuzhou, China), as described previously [13 (link)]. N-cadherin and vimentin were stained using a SPlink Detection kit (ZSGB-BIO, Beijing, China) with rabbit monoclonal anti-N-cadherin (Epitomics, Burlingame, CA, 1:500 dilution) and anti-vimentin (Epitomics, 1:250 dilution) antibodies. A negative control was obtained by replacing the primary antibody with normal rabbit IgG. The expression was recorded as negative if no staining was observed in any array core from the same tissue sample; otherwise, expression was recorded as positive.
7
Western Blot Analysis of EMT Markers
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After the protein extraction and quantification by the Radio Immunoprecipitation Assay (RIPA) lysis buffer (KeyGen, Nanjing, China) and BCA Protein Assay Kit (Takara) respectively, the mixture of proteins (40 µg per sample) and loading buffer (Takara) was loaded on 10% sodium dodecyl sulfate polyacrylamide gel to conduct the electrophoresis for 2 h. Then, the separated proteins on the gel were transferred onto the polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and blocked using 5% non-fat milk (Beyotime). Afterwards, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibody for 1 h at indoor temperature, followed by the detection of the SignalFire™ Plus ECL Reagent (Cell Signaling Technology (CST), Boston, MA, USA). The protein bands were observed by ImageLab software version 4.1 (Bio-Rad Laboratories, Hercules, CA, USA) and the signal levels were analyzed as described earlier.23 (link) The antibodies used in this report were listed as follows: anti-E-cadherin (CST, #3195, 1:1000), anti-N-cadherin (CST, #4061, 1:1000), anti-Vimentin (CST, #5741, 1:1000), anti-KLK4 (Abcam, Cambridge, UK, ab181402, 1:1000), internal control anti-β-actin (CST, #4970, 1:1000), and goat anti-rabbit IgG/HRP-linked secondary antibody (CST, #7074, 1:3000).
8
HCC Cell Line Culturing and Characterization
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The HCC cell lines used in this study included Huh7 cells and LM3 cells. Because the PYCR1 expression of these two cells were higher than the other cells (Figure S1 ). The HCC cell lines were obtained from the China Center for Type Culture Collection (Wuhan, China). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and maintained in a humidified incubator with 5% CO2 at 37 °C.
The following antibodies were used in immunoblotting and immunofluorescence studies: anti-β-actin (HRP-60008; Proteintech Group, China), anti-PYCR1 (131081; Proteintech Group), anti-E-cadherin (PA5-19479; ThermoFisher Scientific, USA), anti-β-catenin (ab32572; Abcam, USA), anti-N-cadherin (ab76011; Abcam), and anti-vimentin (ab92547; Abcam).
Reagents and materials used for the analysis of cell proliferation, migration, and invasion and for RNA-seq included Cell Counting Kit‑8 (CCK‑8; CK04, Dojindo Molecular Technologies, Inc., Rockville, MD, USA), Transwell plates (8 μm pore size; Corning, Inc.), RNA Nano 6000 assay kit (Agilent Technologies, CA, USA), and NEB Next® Ultra™ RNA library prep kit for Illumina® (#E7530L; New England Biolabs, USA).
The following antibodies were used in immunoblotting and immunofluorescence studies: anti-β-actin (HRP-60008; Proteintech Group, China), anti-PYCR1 (131081; Proteintech Group), anti-E-cadherin (PA5-19479; ThermoFisher Scientific, USA), anti-β-catenin (ab32572; Abcam, USA), anti-N-cadherin (ab76011; Abcam), and anti-vimentin (ab92547; Abcam).
Reagents and materials used for the analysis of cell proliferation, migration, and invasion and for RNA-seq included Cell Counting Kit‑8 (CCK‑8; CK04, Dojindo Molecular Technologies, Inc., Rockville, MD, USA), Transwell plates (8 μm pore size; Corning, Inc.), RNA Nano 6000 assay kit (Agilent Technologies, CA, USA), and NEB Next® Ultra™ RNA library prep kit for Illumina® (#E7530L; New England Biolabs, USA).
9
Western Blot Analysis of Signaling Pathways
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Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Non-specific protein interactions were blocked by incubation with 3% non-fat milk in tris-buffered saline with Tween-20 (TBST) at 4°C for 1 h. Subsequently, the membranes were incubated for 2 h with antibodies against GRB2 (1:1,000, Abcam, Cambridge, UK), anti-p-AKT (Abcam, Cambridge, UK), anti-AKT (Abcam, Cambridge, UK), anti-ERK1/2 (Cell Signaling Technology CST, Danvers, MA, USA), anti-p-ERK1/2 (CST), anti-N-cadherin (CST), anti-E-cadherin (CST), anti-vimentin (CST) and anti-GAPDH (Abcam, Cambridge, UK). Next, the membranes were incubated with fluorescent secondary antibodies. GAPDH was used as the endogenous control.
10
Quantifying E-cadherin and N-cadherin in HCC
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Hepatocellular carcinoma cells fixed in 4% formaldehyde were treated with pepsin and subjected to dehydration through EtOH. After permeabilization in Triton X‐100 (Sigma‐Aldrich Co., St. Louis, MO, USA), samples were blocked by goat serum and then incubated with anti‐E‐cadherin and anti‐N‐cadherin (Abcam, Cambridge, MA, USA) overnight at 4 °C. After washing off primary antibodies, cells were incubated for 1 h with appropriate secondary antibody. Cell nuclei were then stained with DAPI. The visualization was conducted using a fluorescence microscope (DMI4000B; Leica Microsystems, Wetzlar, Germany).
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