Enhanced ecl detection kit

Cells were washed twice with PBS and total cellular proteins were extracted using a radioimmunoprecipitation assay (0.5% Nonidet P-40, 10 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% sodium dodecyl sulfate). Whole cell lysates were sonicated, boiled at 95°C for 5 min and then chilled on ice for 10 min. The relative level of protein expression was determined using specific antibodies. Antibodies used for Western blots were: rabbit antibodies against Syntaxin 2 (ab12369, Abcam), α-SMA (Sigma), phospho-FAK (Tyr-397, Biosource International, Camarillo, CA), ERK 1/2, phospho-ERK 1/2 (Thr202/Tyr204), MMP-3 and TIMP-1(Cell Signaling Technology), GAPDH (ab70699, Abcam); and mouse monoclonal antibodies against FAK (Upstate Biotechnology, NY). The immunoreactions were visualized using an enhanced ECL detection kit (Amersham Pharmacia Biotech), exposed to X-ray film and quantified using a video documentation system (Gel Doc 2000, Bio-Rad).

Freshly collected liver tissue from HCC and noncancerous control was snap frozen immediately in liquid nitrogen. Tissues were homogenized in ice-cold TRIS-EDTA buffer (PH 7.4) with protease inhibitors (Sigma-Aldrich, USA). Protein was estimated by Bradford method using Pierce BCA protein assay kit (Thermo Fisher scientific, USA).. Equal amounts of protein extract were denatured and separated on 4-12% NuPAGE Bis-Tris Gels and transferred on to PVDF membranes (Invitrogen, UK), which were then probed with rabbit anti-ZO-1 (ThermoFisher Scientific, USA) and rabbit anti-β-actin (abcam, USA) with HRP-conjugated secondary antibody. The bands were visualized using an enhanced ECL detection kit (Amersham, UK) and quantified by densitometry.