Western Blot Analysis of Hedgehog Pathway

Total protein extracts were harvested and subjected to WB as described previously [23 (link)]. Primary antibodies against the following proteins were used for WB: GLI2 (ab26056; Abcam), PTCH1 (ab55629; Abcam), FOXM1 (sc-376,471; Santa Cruz Biotechnology, CA, USA), TPX2 (12,245; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MAB374; Millipore, Billerica, MA, USA). This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, namely normal goat anti-mouse IgG (31,430; Thermo Scientific Pierce) or normal goat anti-rabbit IgG (31,460; Thermo Scientific Pierce), and the membranes were probed with SuperSignal™ West Femto Maximum Sensitivity Substrate ECL (34,095; Thermo Fisher Scientific Inc). The immunoblot films were digitalized with Epson V700 scanner, and intensity of major bands were quantitated using Image J (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least thrice.