Cells cultured on cover slide were washed with PBS and fixed in acetone for 10 min at 4 °C, followed by being incubated with primary antibodies: anti-Ki67 (mouse, 1:100; Sigma-Aldrich, USA), anti-BrdU (mouse, 1:100; Sigma-Aldrich, USA), and β-catenin (Rabbit, 1:100; Boster, Wuhan, China), anti-E-cadherin (Rabbit, 1:100; Boster, Wuhan, China), relevant secondary antibodies and counter-stained with DAPI (1:1000; Sigma-Aldrich, USA). Fluorescence was detected by fluorescence microscopy (Nikon, Tokyo, Japan).
Anti e cadherin
Manufactured by Boster Bio
Sourced in China
Anti-E-cadherin is a primary antibody used in immunochemistry applications. It specifically binds to the E-cadherin protein, which is involved in cell-cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (2 mentions)
β catenin, by Boster Bio (1 mentions)
Anti brdu, by Merck Group (1 mentions)
Anti ki67, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti aeg 1, by Abcam (1 mentions)
Anti mmp9, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Anti pi3k p85, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
4 protocols using anti e cadherin
1
Immunofluorescence Assay for Cell Markers
2
Western Blot Protein Expression Analysis
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Total proteins of cells were extracted according to the protocol of protein extraction kit (KeyGen, China), and the protein concentration of each sample was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Equivalent quantities of protein were separated by 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 10% defatted milk (10% BSA for p‐protein) and then incubated with the appropriate primary antibody overnight at 4°C. They were next washed and incubated with the corresponding horseradish peroxidase (HRP)‐conjugated secondary antibody for 1 h. Bound secondary antibody was visualized using an enhanced chemiluminescence (ECL) system (Pierce Biotechnology). The primary antibodies used were anti‐GAPDH (1:1000, Cell Signal Technology, Danvers, MA), anti‐AEG‐1 (1:10000, Abcam, USA), anti‐E‐cadherin(1:200 Boster bio., Shanghai, China), anti‐β‐catenin (1:1000, Cell Signal Technology), anti‐MMP2 (1:1000, Abcam, USA), and anti‐MMP9 (1:1000, Cell Signal Technology). The results were normalized to GAPDH to correct for loading.
3
Western Blot Analysis of Signaling Proteins
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Anti‐ERRα Ab was obtained from Novus Biologicals, anti‐Nectin‐4 Ab was obtained from Abcam, anti‐Lamin B1 Ab was purchased from Bosterbio, and anti‐E‐cadherin, anti‐Vimentin, anti‐PI3K p85, anti‐p‐PI3K p85α (phospho‐Y607), anti‐AKT, anti‐p‐AKT (Ser473), and β‐actin Abs were acquired from Cell Signaling Technology. Proteins from cells were separated by SDS‐PAGE and then transferred to PVDF membranes (Millipore). We blocked the membranes with 5% skimmed milk (5% BSA for the testing of p‐PI3K and p‐AKT) for 1 hour. The membranes were incubated with diluted primary Abs at 4°C for 12 hours. Afterward, the membranes were incubated with the secondary Ab for 1 hour at room temperature. Enhanced chemiluminescence (Millipore) was used to detect the targeted signal. β‐actin and Lamin B1 served as the loading controls for total protein and nucleoprotein, respectively. All assays were carried out in triplicate.
4
Protein Extraction and Western Blotting
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Total protein was extracted in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were quantified by bicinchoninic acid (BCA) assay (Beyotime Institute of Biotechnology). Western blotting was performed as previously described. 22 The following primary antibodies were used: anti-MMP-2 (1:500; Bioworld Technology, Atlanta, GA, USA), anti-MMP-9 (1:500; Bioworld Technology), anti-E-cadherin (1:300; Boster, Wuhan, China), anti-Vimentin (1:200; Boster), anti-phosphorylated AKT (1:500; Cell Signaling Technology, Beverly, MA, USA), anti-AKT (1:1000; Cell Signaling Technology) and antiβ-actin (1:300; Zhongshan Golden Bridge, Beijing, China). Protein bands were quantified by densitometry using Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to β-actin or total AKT.
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