Western blot was done according to our previous study.17 (link) HCC were treated with CTPG for 24 hours. After washing with ice-cold PBS
twice, all adherent and floating cells were collected and lysed in RIPA Lysis
Buffer (Beijing ComWin Biotech Co., Ltd) for 20 minutes on ice. After
centrifugation at 12 000 rpm 4°C for 10 minutes, protein concentration was
determined using a Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, USA)
according to the manufacturer’s instructions. The same concentration of proteins
was separated by 12% SDS-PAGE and transferred to PVDF membranes. After washing
with PBST buffer (PBS with 0.05% Tween-20), the membranes were blocked with 5%
non-fat milk at 37°C for 1 hour, and then incubated with the primary antibodies
(Cell Signaling Technology, MA, USA) at proper dilutions overnight at 4°C. After
washing 3 times with PBST, the membrane was incubated with the corresponding
HRP-conjugated secondary antibodies (eBioscience) for 2 hours at 37°C. The
target proteins were detected using ECL assay kit (Beyotime). Grayscale scanning
data were obtained by Image J.