Protein extraction and western blotting analysis were performed using previously standard procedures (17 (link)). The following antibodies were used for western blotting: anti-E-cadherin antibody(#20874-1-AP Proteintech, China), anti-N-cadherin antibody(#22018-1-AP Proteintech, China), anti-ZEB1 antibody(#66279-1-Ig Proteintech, China), anti-Vimentin antibody(10366-1-AP Proteintech, China), anti-SOX2 antibody(#11064-1-AP Proteintech, China), anti-GAPDH antibody(#10494-1-AP Proteintech, China), and anti-PD-L1 antibody(#66248-1-Ig Proteintech, China).
Anti n cadherin antibody
Manufactured by Proteintech
Sourced in United States
The Anti-N-cadherin antibody is a laboratory reagent designed for the detection and analysis of N-cadherin, a cell adhesion molecule. This antibody can be used in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of N-cadherin in biological samples.
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Lab products found in correlation
Anti e cadherin antibody, by Proteintech (4 mentions)
Anti vimentin antibody, by Proteintech (4 mentions)
Anti gapdh antibody, by Proteintech (2 mentions)
Anti zeb1 antibody, by Proteintech (1 mentions)
Anti sox2 antibody, by Proteintech (1 mentions)
Primary antibody dilution buffer, by Meilun (1 mentions)
Fd8030, by Fudebio (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Fdm007, by Fudebio (1 mentions)
Fd1001, by Fudebio (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Ripa lysis buffer, by Meilun (1 mentions)
Anti ki67 antibody, by Proteintech (1 mentions)
Anti cd163 antibody, by Proteintech (1 mentions)
Anti vegfa antibody, by Proteintech (1 mentions)
Anti cd8 alpha antibody, by Abcam (1 mentions)
6 protocols using anti n cadherin antibody
1
Protein Extraction and Western Blotting
2
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA Lysis Buffer (MA0151, Dalian Meilun Biotechnology Co., Ltd., China) containing a protease inhibitor cocktails (FD1001, Fudebio, Hangzhou, China) on ice. Equal amounts of total protein from different samples were separated by SDS-PAGE gels at 100 V for 1.5 h and transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ) at 280 mA for 1.5 h. Then, the membranes were blocked with 5% skim milk powder in TBST for 1 h at room temperature and treated with specific primary antibodies overnight at 4 °C. The next day, the membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China). Each band was detected using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). Anti-GAPDH, anti-CREB3, anti-Bcl-2, anti-mmp2, anti-Vimentin and anti-E-cadherin antibodies were purchased from Abcam (Cambridge, UK). The anti-c-Jun, anti-cleaved-caspase 3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA); anti-mmp9 antibody was purchased from ABclonal (Boston, MA, USA); and anti-N-cadherin antibody was purchased from Proteintech (Chicago, USA). Primary Antibody Dilution Buffer was purchased from Dalian Meilun Biotechnology Co., Ltd. (MB9881, Dalian, China).
3
Immunohistochemical Analysis of Cancer Biomarkers
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Thirty-six representative samples from C1 (N = 18) and C2 (N = 18) were selected for tissue microarray (TMA) construction, the TMA is prepared as previously described43 . For immunohistochemistry staining, the sections were stained using anti-Vimentin antibody (Proteintech, 1:2000), anti-Ki67 antibody (Proteintech, 1:5000), anti-Cyclin D antibody (Proteintech, 1:1500), anti- E-cadherin antibody (Proteintech, 1:2000), anti- N-cadherin antibody (Proteintech, 1:200), anti-ADH1A antibody (Abcam, 1:500), anti-CYP3A4 antibody (Proteintech, 1:200), anti-CD34 antibody (Proteintech, 1:1000), anti-VEGFA antibody (Proteintech, 1:200), anti-CD8-alpha antibody (Abcam, 1:500), anti-CD163 antibody (Proteintech, 1:1000). Images were captured by MoticEasyScan (Motic).
4
Western Blot Analysis of Cellular Markers
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Protein extraction and western blotting were performed as described previously [46 (link)]. The antibodies were as follows: anti-α-SMA (abcam, ab5694), Vimentin (Proteintech, 10366-1-AP), anti-E-cadherin antibody (proteintech, 20874-1-AP), anti-CD63 antibody (santa cruze, sc-5275), anti-CD81 antibody (proteintech, 27855-1-AP), anti-TSG101 antibody (Santa Cruz, California, United States, sc-7964), anti-RNF43 antibody (bioss, bs-24331R), anti-β-catenin antibody (proteintech, 51067-2-AP), anti-ALDH1A1 antibody (proteintech, 15910-1-AP), anti-OCT4 antibody (proteintech, 11263-1-AP), anti-N-cadherin antibody (proteintech, 66219-1-Ig), and anti-α-tubulin (abcam, ab7291). Protein levels were normalized to α-tubulin and analyzed by Image J software.
5
Western Blot Analysis of EMT Markers
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Cells were lysed in buffer containing 50 mmol·L−1 Tris/HCl, pH 8.0, 150 mmol·L−1 NaCl, 0.02% NaN3, 0.1% SDS, 100 mg·L−1 phenylmethylsulfonylfluoride, 1 mg·L−1 aprotinin and 1% Triton. Cell extract was separated by SDS/PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h in TBST (10 mmol·L−1 Tris/HCl, pH 7.4, 150 mmol·L−1 NaCl, 0.05% Tween‐20) containing 5% BSA, incubated with the primary antibodies anti‐E‐cadherin antibody (Proteintech), anti‐N‐cadherin antibody (Proteintech), anti‐vimentin antibody (Proteintech, Rosemont, IL, USA), anti‐α‐SMA antibody (Sigma), anti‐FAP antibody (Abcam), anti‐GDF10 antibody (R&D) at 4 °C overnight, followed by incubation with secondary antibodies. Bands were visualized with an enhanced chemiluminescence reaction (Millipore Corp., Billerica, MA, USA). GAPDH was used as the loading control. Protein bands were captured and analyzed using lane 1D software (Sage Creation Science Co., Beijing, China).
6
Quantitative Protein Expression Analysis
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First, RIPA buffer (NaCl 150 mM, 50 mM Tris (pH = 7.5), Triton X-100 1% (v/v), Na-deoxycholate 0.05% (w/v), sodium deoxycholate 0.01% (w/v), 1% cocktail (v/v), 1% phosphatase inhibitors (v/v)) was used to lyse the tissues or cells. After centrifugation, the Bradford assay was used to measure the protein concentrations. The prepared protein samples were separated by 10% or 12% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBST and then incubated with anti-N-cadherin antibody (Proteintech, 22018-1-AP) (1:1000), anti-vimentin antibody (Proteintech, 10366-1-AP) (1:1000), anti-RAI14 antibody (Proteintech, 17507-1-AP) (1:1000), or anti-GAPDH antibody (Proteintech, 60004-1-Ig) (1:5000) overnight at 4 °C. Then, after washing three times with PBST, the membranes were incubated with the secondary antibody at room temperature for 1 h. Band images were detected using Immobilon Western HRP Substrate (Millipore).
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