Immunoblotting was exercised to examine the expression of Hh signaling pathway-related markers in HCC cells according to standard protocols as described previously.18 (link) Protein was harvested using radio immunoprecipitation assay lysis buffer, and a bicinchoninic acid assay was applied to determine the protein concentration. Next, we separated equal amount of protein using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred the protein to a polyvinylidene difluoride membrane. After blocking with 1.5% skimmed milk for 1 h at room temperature, membranes were incubated with the primary antibodies at 4°C overnight and with horseradish peroxidase-conjugated secondary antibodies. Membranes were developed using a chemiluminescent substrate. The antibodies were used as follows: Gli1 (1:200, ab49314, Abcam), Gli2 antibodies (1:500, ab26056) and Gli3 antibodies (1:500, ab6050) and the secondary antibodies (1:50,000, ab7090).