The total protein from both morusin-treated HCC cells and tumor tissues was prepared as previously described.23 (link) Equal amounts of protein from each sample were subjected to 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk at room temperature for 1 h and then incubated with the primary antibody overnight at 4°C. Primary antibodies were rabbit anti-STAT3, rabbit anti-p-STAT3 (Tyr705), rabbit anti-p-STAT3 (Ser727), rabbit anti-IL-6, rabbit anti-Bcl-2, rabbit anti-Bax, rabbit anti-active caspase-3, rabbit anti-MMP2, rabbit anti-MMP9, rabbit anti-VEGF and rabbit anti-VEGFR2, and rabbit anti-β-actin (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). Subsequently, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China) at 1:6,000 dilution. Immune complexes were detected by Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA) and a Western blot analysis system (Universal Hood II; Bio-Rad Laboratories Inc., Hercules, CA, USA).