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 e cadherin antibody
Manufactured by Proteintech
Sourced in United States, China
The Anti-E-cadherin antibody is a laboratory tool used to detect and study the E-cadherin protein. E-cadherin is an essential cell-cell adhesion molecule found in epithelial tissues. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and analyze the expression and localization of E-cadherin in biological samples.
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Lab products found in correlation
Anti vimentin antibody, by Proteintech (5 mentions)
Anti n cadherin antibody, by Proteintech (4 mentions)
Anti gapdh antibody, by Proteintech (2 mentions)
Anti vimentin antibody, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by Merck Group (2 mentions)
Anti n cadherin antibody, by Cell Signaling Technology (2 mentions)
Anti smad3 antibody, by Cell Signaling Technology (2 mentions)
Anti p smad3 antibody, by Cell Signaling Technology (2 mentions)
Anti ptbp1 antibody, by Cell Signaling Technology (2 mentions)
Anti ptbp2 antibody, by Abcam (2 mentions)
Anti ptbp3 antibody, by Merck Group (2 mentions)
Anti hnrnpk antibody, by Abcam (2 mentions)
Anti hnrnpm antibody, by Merck Group (2 mentions)
Anti fubp3 antibody, by Abcam (2 mentions)
Anti cpsf2 antibody, by Abcam (2 mentions)
Anti tgfβ2 antibody, by Abcam (2 mentions)
Anti p smad2 antibody, by Cell Signaling Technology (2 mentions)
Anti smad2 antibody, by Cell Signaling Technology (2 mentions)
Anti smad5 antibody, by Abcam (2 mentions)
Anti id2 antibody, by Abcam (2 mentions)
12 protocols using anti e cadherin antibody
1
Protein Extraction and Western Blotting
2
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).
3
Western Blotting Analysis of EMT Markers
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Western blotting analysis was performed using the standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-PTBP2 antibody (Abcam, Cambridge, UK), anti-PTBP3 antibody (Sigma-Aldrich, St. Louis, MO, USA), anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody (Cell Signaling Technology), anti-SMAD2 antibody (Cell Signaling Technology), anti-p-SMAD3 antibody (Cell Signaling Technology), anti-SMAD3 antibody (Cell Signaling Technology), anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology) and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit (Pierce, Rockford, IL).
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 Regulators
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The Western blotting analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-PTBP2 antibody (Abcam, Cambridge, UK), anti-PTBP3 antibody (Sigma-Aldrich, St. Louis, MO, USA), anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody (Cell Signaling Technology), anti-SMAD2 antibody (Cell Signaling Technology), anti-p-SMAD3 antibody (Cell Signaling Technology), anti-SMAD3 antibody (Cell Signaling Technology), anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology) and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with a goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit (Pierce, Rockford, IL).
6
Gastric Mucosal Protein Expression Analysis
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The gastric mucosal tissues of rats were weighed, and then placed into the RIPA lysis buffer (BN25012, Bairuiji, China) containing cell lysis magnetic beads prepared in advance. The samples were ground with a tissue crushing homogenator and the supernatant was retained for BCA assay and further preparation for SDS-PAGE. The separated bands were transferred onto nitrocellulose membrane (Merck Millipore, United States). The membrane was blocked with 5% skim milk at room temperature for 30 min and then incubated with primary antibodies overnight at 4°C. The primary antibodies including: Anti-EGFR antibody (#4267, CST), Anti-PI3 Kinase antibody (#67071-1-Ig, Proteintech), Anti-p-AKT antibody (#4060, CST), Anti-AKT antibody (#4691, CST), Anti-N-cadherin antibody (#14215S, CST), Anti-E-cadherin antibody (#60335-1-Ig, Proteintech) Anti-β-Catenin antibody (#610154, CST), Anti-Vimentin antibody (#60330-1-Ig, Proteintech), Anti-GAPDH antibody (#5174, CST). After secondary antibody incubation, the membranes were washed 3 times and detected by West Pico PLUS Chemiluminescence substrate (#34577, Thermo, United States) and images were obtained using the ChemiScope 3,000 detection system (Qingxiang, Shanghai, China).
7
Purification and Characterization of rhMG53
8
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).
9
Protein Extraction and Western Blot
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Total proteins were extracted from the cells with different treatments using RIPA lysis buffer (cat no. P0013B, Beyotime Biotechnology), and the concentrations of the total proteins were determined using a BCA assay kit (cat no. AR0146, Boster Bioengineering Co., Ltd., Wuhan, China). The protein samples (20 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, and then blocked with 5% skim milk at 37 °C for 2 h. After washing with PBST (1 mL Tween-20 in 1000 mL 1 × PBS), the membranes were respectively incubated with anti-caspase3 antibody (1:1000, cat no. 19677-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), anti-E-cadherin antibody (1:2000, cat no. 20874-1-AP, Proteintech Group, Inc.), anti-TIMP3 antibody (1:1000, cat no. 10858-1-AP, Proteintech Group, Inc.), and anti-GAPDH antibody (1:10000, cat no. 60004-1-AP, Proteintech Group, Inc.) overnight. The PVDF membrane was washed 3 times with TBST and incubated with the secondary antibody (goat anti-rabbit IgG-HRP, 1:5000, cat no. 111-035-003, Jackson ImmunoResearch, USA) at room temperature for 2 h. After washing 3 times with TBST, the protein-containing membrane was detected using the ECL chemiluminescence detection system (model no. T4600, Shanghai Tianneng Technology Co., Ltd., Shanghai, China).
10
Immunofluorescence Assay of Ishikawa Cells
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Different groups of Ishikawa cells cultured in chamber slides were fixed in 4% paraformaldehyde, followed by permeabilization and blocking. Cells were then incubated with respective primary antibodies, including anti-vimentin antibody (1:2500, Proteintech, Hubei, China), anti-E-cadherin antibody (1:2000, Proteintech), overnight at 4°C. Cy3-labeled goat anti-rabbit IgG (1 μg/mL, Sigma) and goat anti-mouse IgG (1 μg/mL, Sigma) were used, respectively, to visualize the signal, and nuclei were stained with DAPI (1 μg/mL, Sigma). The cell pictures were observed under an inverted fluorescence microscopy imaging system (Olympus).
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