Ecl western blot system

After treatments, cells were washed and lysed as previously described [34 (link)]. Twenty micrograms of total protein extract were separated by 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare Milano, Italy) in a cooling system at 100 V for 1 h. Membranes were saturated for 1 h at room temperature with 0.1% Tween-20 (Sigma-Aldrich), 5% dry milk (Bio-Rad Laboratories, Hercules, CA) in PBS. Membranes were then incubated with an antibody against human tissue non-specific alkaline phosphatase (Abcam, Cambridge, UK) diluted 1:10,000, overnight at 4°C, washed several times and incubated with peroxidase-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:10,000, for 1 h at room temperature. Specific bands were then detected by ECL Western blot system (GE Healthcare). Antibody against GAPDH (Santa Cruz Biotechnology) were used to perform a normalizing control. Densitometry of bands was performed with ImageJ software [http://rsbweb.nih.gov/ij/download.html]. Molecular sizes were evaluated referring to protein molecular weight standards (Bio-Rad Laboratories). Each treatment and analysis was performed at least in triplicate separate experiments.

Total protein was extracted using 400 μl protein extraction fluid (HuaTe Sheng, China) with 40 μl (10 mM) phenylmethanesulfonyl fluoride (PMSF) according to the supplier’s instructions. The concentration of the total protein was determined using BCA Protein Assay Kit (Pierce Chemical Co, USA). 60 μg of denatured total protein of each sample per lane were resolved by SDS-PAGE (15 % acrylamide), and transferred to a nitrocellulose membrane (Millipore, USA) using a Semi-DRY Transfer cell (Bio-Rad Laboratories Inc, Hercules, CA, USA). The membrane was blocked with 5 % defatted milk (in 25 mM Tris, pH 8.0, 125 mM NaCl, 0.1 % Tween 20) for 1 h at room temperature then incubated with 1:1000 diluted anti-CLDN6 (Santa Cruz Biotechnologies) antibody at 4 °C overnight. After washing three times with PBS, the membrane was incubated with a horseradish peroxidase conjugated anti-goat IgG (1:1000, Santa Cruz Biotechnologies) secondary antibody at room temperature for 1 h. After washing, the immunoreactive bands were visualized using an ECL western blot system (GE, USA) and exposed to X-ray film (Estman Kodak). Anti-β-actin (Santa Cruz Biotechnologies) antibody was used as the endogenous control.