Mouse monoclonal anti vimentin

RIPA lysis buffer (Auragene, Changsha, China) was used to extract protein from indicated cells. Bradford Protein Assay Kit (Beyotime, Shanghai, China) was used to measure the protein concentration. A total of 50 µg of protein was separated on 10% SDS-PAGE gels and blotted onto 0.22-µm nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 2 h and incubated with primary antibodies (rabbit polyclonal anti-E-cadherin, cat No. ab15148, 1:1,000 dilution), rabbit polyclonal anti-fibronectin (cat No. ab2413, 1:1,000 dilution), mouse monoclonal anti-vimentin (cat No. ab8978, 1:500 dilution) from Abcam (Cambridge, UK), and mouse monoclonal anti-β-actin (cat No. 60008-1-Ig, 1:5,000 dilution) from Proteintech (Wuhan, China) overnight at 4°C. The membranes were washed with Tris-buffered saline containing 0.1% Tween 20 (TBST), and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:2,000; goat anti-mouse, 1:5,000; Auragene) for 1 h at 37°C. Enhanced chemiluminescence reagent (Merck Millipore, Germany) was used to detect the signal on the membrane. The data were analyzed via densitometry using Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) and normalized to the expression of the internal control (β-actin).

The protein was extracted by radioimmunoprecipitation assay lysis buffer from indicated cells, and BCA Protein Assay Kit (Thermo Scientific) was used to measure the protein concentration. After being separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, the protein was transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk and incubated with primary antibodies (rabbit polyclonal anti-E-cadherin [1:1,000], mouse monoclonal anti-vimentin [1:1,000], rabbit polyclonal anti-fibronectin [1:1,500], and rabbit monoclonal anti-beta Catenin [1:2,000] from Abcam, Cambridge, UK; goat polyclona anti-ZEB1 [1:500] and mouse monoclonal anti-ZEB2 [1:500] from Santa Cruz Biotechnology Inc., Dallas, TX, USA; mouse monoclonal anti-GAPDH [1:5,000] from Sigma-Aldrich Co., St Louis, MO, USA) overnight at 4°C. The membranes were washed with TBST, and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescence reagent was used to detect the signal on the membrane.