Anti β actin antibody

HCC70 and MDA-MB-231 cells were treated or not with TNF-α (50 ng/mL) for 18 h. Total proteins were extracted with RIPA buffer. Proteins were separated by 7.5% denaturing SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with the anti-VCAM-1 antibody (1/2000; Rabbit anti-VCAM-1, ab134047 Abcam®, Cambridge, UK). As a loading control, the membrane was probed with an anti-β-actin antibody (1/10,000; Beckton Dickinson). Revelation was assessed using the chemiluminescence ECL kit (BioRad). Bands were quantified by densitometry using ImageJ software.

Protein lysates were prepared from different experimental groups of cells and tissues and quantified with the bicinchoninic acid (BCA) kit (Wuhan Boster Biological Technology Co., Ltd. Wuhan, China) according to the manufacturer’s instructions. Then, equal amounts of protein (30 µg) were separated by 10% polyacrylamide gel electrophoresis (PAGE) (Wuhan Boster Biological Technology Co., Ltd. Wuhan, China) at 100V for 2 h and transferred onto PVDF membranes at 100V for 1 h. Then, after blocking the membrane with 5% skimmed milk in 1X TBST buffer, the blots were incubated overnight at 4°C with anti-TLR4 (ab30667) and anti-NF-κB (ab31432) antibodies (both from Abcam Inc., Cambridge, MA, USA; 1:1000 dilution) as well as anti- β-actin antibody (Becton, Dickinson and Company, NJ, USA; 1:3000) as internal control. Then, after washes with 1X TBST, the blots were incubated at room temperature with HRP-conjugated secondary antibodies for 2 h. After washing, the blots were developed with ECL chemiluminescence reagent and imaged with a Gel Doc EZ Imager (Bio-Rad Laboratories, Inc. CA, USA). The grey values of the protein bands were quantified with the Image J software.

The proteins were extracted from the whole cell lysates using RIPA cell lysis buffer and the protein concentration was determined. In total, 20 μg of the extracted total cellular protein from each sample were separated via SDS-PAGE, and transblotted onto EMD Millipore Immobilon™-P PVDF Transfer Membranes (EMD Millipore Cat No.: IPVH00010, USA). Western blot analyses were conducted with the following antibodies: mouse monoclonal anti-CD109 (Cat. No. 556039, BD Biosciences), anti-fibronectin (Cat. No.610078, BD Biosciences), anti-β-actin antibodies (Cat. No. sc-47778, Santa Cruz Biotechnology), rabbit polyclonal anti-Snail (Cat. NO. ab5351, abcam), anti-twist (Cat. No. ab505181; abcam), anti-Sox2(Cat. No: ab137385, abcam), and anti-Slug (Cat. No. 9585 S Cell signaling).