Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors. Total protein was quantified using a BCA kit (Wuhan Boster Biological Technology Co., Ltd.), according to the manufacturer's protocols, and 20 μg protein/lane was separated via SDS-PAGE on a 10% gel. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% non-fat milk for 1 h at room temperature. The membranes were then incubated with the following primary antibodies: Anti-E-cadherin (1:1,000; cat. no. 14472; Cell Signaling Technology, Inc.), anti-N-cadherin (1:1,000; cat. no. 13116; Cell Signaling Technology, Inc.), anti-vimentin (1:1,000; cat. no. 5741; Cell Signaling Technology, Inc.), anti-GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) and anti-RYBP (1:500; cat. no. sc-374235; Santa Cruz Biotechnology, Inc.). Following the primary antibody incubation, the membrane was washed three times with TBS with 2% Tween-20 and incubated with HRP-conjugated anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) secondary anti-bodies (1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using Immobilon Western Chemiluminescent HRP substrate (EMD Millipore) and an ImageQuant™ LAS 4000 mini-imaging system (Cytiva). Semi-quantification was performed using ImageJ (v1.8.0).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.