Phospho c met

HepG2 cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Cell lysates (20 μg protein/lane) were loaded and separated on gradient polyacrylamide gels and then transferred to polyvinylidene difluoride membranes by electroblotting (Millipore Corp., Boston, MA, USA). Following blocking with 5% non-fat milk containing 0.3% Tween 20 for 1 h, the membranes were incubated overnight with primary antibodies at 4°C, including anti-hSulf-1 (1:250), -stat3 (1:500), -phospho-stat3 (1:500), -phospho-c-met (1:500), -bcl-2 (1:1000) and -cyclin D1 (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were washed three times with Tris-buffered saline containing Tween 20 and membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (R&D Systems China Co., Ltd., Shanghai, China) at 4°C for 1 h. Subsequently, membranes were exposed to enhanced chemiluminescent reagents for detection of protein bands. β-actin was used as an internal control.