Total protein was extracted from SKOV3 and 3AO cells with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. The same amount of protein in each group was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then the proteins were transferred to the nitrocellulose (NC) membrane (Pall Life Sciences, Port Washington, NY, USA). After that, the membrane was blocked with 5% skim milk for 1.5 h and then incubated with the corresponding specific primary antibody at 4 °C overnight. Then, the secondary antibody (1:5000) was employed to incubate the membrane at room temperature for 1 h. Ultimately, electrochemical luminescence (ECL) kit (Beyotime, Shanghai, China) was used to detect the immunoreactivity. The primary antibodies used in this work included anti-E-cadherin antibody (Abcam, ab197751, 1:2000), anti-vimentin antibody (Abcam, ab92547, 1:1000), anti-β-catenin antibody (Abcam, ab16051, 1:1000), anti-c-Myc antibody (Abcam, ab32072, 1:2000), anti-cyclin D1 antibody (Abcam, ab16663, 1:2000) and anti-GAPDH antibody (Abcam, ab9484, 1:4000).
Ab197751
Manufactured by Abcam
Sourced in United States
Ab197751 is a laboratory equipment item produced by Abcam. It serves as a core functional component in various experimental setups. The detailed specifications and intended applications of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab92547, by Abcam (3 mentions)
Ab16051, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab9484, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Nc membrane, by Pall Corporation (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab32072, by Abcam (1 mentions)
Ab174850, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab202030, by Abcam (1 mentions)
Ab181557, by Abcam (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ab97959, by Abcam (1 mentions)
Sc 373800, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ab197751
1
Western Blot Analysis of EMT Markers
2
Extraction and Analysis of JAM-A, CD138, and E-cadherin from Serum and Tumor Tissue
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For serum samples, blood samples were clotted for 2 h at room temperature and centrifuged at 2000g for 20 min. For TIF, tumor tissue was put on 10 µm Nylon Net filter (NY1002500, Sigma), centrifuged at 60 g for 10 min and collect fluid. Samples were then analyzed for mouse JAM-A (ab277080, Abcam), mouse CD138 (NBP2-76610, Novus) and mouse E-cadherin (ab197751, Abcam) according to manufacturer’s instruction.
3
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using the RIPA buffer (Beyotime, Nantong, China), and then, we tested the protein concentration by bicinchoninic acid protein assay (BCA). Equal amounts of proteins were isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA, USA). After blocking with 5% nonfat dried milk for 1h, the membrane was incubated with the primary antibody against NDRG2 (1 : 1000; ab174850, Abcam, Cambridge, USA), E-cadherin (1 : 1000; ab197751, Abcam), N-cadherin (1 : 1000; ab256744, Abcam), vimentin (1 : 1000; ab92547, Abcam), p-PI3K (1 : 1000, 17366, Cell Signaling, San Jose, CA, USA), PI3K (1 : 1000, 3821, Cell Signaling), p-AKT (1 : 1000, 5197, Cell Signaling), AKT (1 : 1000, 4685, Cell Signaling), and GAPDH (1 : 3000, Cell Signaling) at 4°C overnight. Subsequently, the membranes were incubated with the secondary HRP-conjugated antibody (Beyotime) for 2 h at room temperature. Finally, the protein signal was determined with the enhanced chemiluminescence (ECL) kit (Pharmacia Biotech, Arlington, USA).
4
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from clinical tissue samples and cancer cells by radio-immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, USA) containing protease inhibitor (Biyuntian, Shanghai) for 30 minutes at 4°C as described previously [16 (link),17 (link)]. Vector and experimental group cells were collected, washed 3 times with ice-cold PBS and then protein was extracted using RIPA lysate buffer containing protease inhibitor (Biyuntian, Shanghai). Denatured proteins (50 ug) were separated using 12% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and blotted onto nitrocellulose membranes (polyvinylidene difluoride). The membranes transferred by cell lysates were subsequently incubated with primary antibodies including: β-catenin (1: 1000; ab16051; Abcam, USA), GSK-3β(1: 1000; ab141295; Abcam), p-GSK-3β (1: 1000; sc-373800; Santa Cruz, USA), E-cadherin (1: 1000; ab197751; Abcam), N-cadherin (1: 1000; ab202030; Abcam), vimentin (1: 1000; ab92547; Abcam), OCT4n (1: 1000; ab181557; Abcam), and SOX2 (1: 1000; ab97959; Abcam). After 24 hours, the membranes were washed with PBS twice and then incubated with horseradish peroxidase-conjugated goat anti-rabbit for 2 hours at 37°C, immunoreactive bands were analyzed with by applying Imaging System (Thermo Fisher Scientific, USA). All assays were repeated thrice.
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