Emodin induces apoptosis in human hepatocellular carcinoma HepaRG cells via the mitochondrial caspase-dependent pathway

Dong, Xiaoxv; Ni, Boran; Fu, Jing; Yin, Xingbin; You, Longtai; Leng, Xin; Liang, Xiao; Ni, Jian

Western blot analysis. HepaRG cells were plated in 6-well plates and treated as described above. The cells were harvested, lysed with RIPA buffer for 30 min on ice. Lysates were centrifuged at 12,000 rpm for 10 min at 4˚C. Total protein concentration was determined using the bicinchoninic acid assay (BCA) protein assay kit. In a parallel experiment, cytosolic and mitochondrial extracts were prepared using the ProteoExtract ® Cytosol/Mitochondria Fractionation Kit (Millipore, Billerica, MA, USA) following the manufacturer's protocol. An equal amount of proteins was separated by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with TBST buffer containing 5% skim milk for 1 h, and then incubated with specific primary antibodies overnight at 4˚C. After three times washing with TBST buffer, the membranes were further incubated with the horseradish peroxidase-conjugated secondary antibodies (cat. no. 3700; Cell Signaling Technology) at room temperature for 1 h (37, 38) . An enhanced ECL detection system was used for visualization of target proteins (iNtRON Biotechnology, Seongnam, Korea).