Extraction of Nuclear and Cytoplasmic Proteins and Western Blotting

For protein analysis, adherent cells were collected with 0.25% trypsin (Gibco; USA) and centrifuged at 1,000 rpm for 5 min. Next, cells were washed twice with PBS and nuclear and cytoplasmic proteins were extracted with the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo, USA). Protein concentration was then determined using the bicinchoninic acid (BCA) protein assay kit (Thermo, USA) according to the manufacturer’s instructions. Both nuclear and cytoplasmic proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.45 μm polyvinylidene difluoride membranes (PVDF, Bio-Rad; USA), and blocked with 1% bovine serum albumin for 1 h. Then, membranes were incubated overnight at 4°C with rabbit anti-Shh monoclonal antibody (1:2000, ab53281; Abcam), rabbit anti-Gli1 polyclonal antibody (1:1000, ab49314; Abcam), mouse anti-NF-κB p65 monoclonal antibody (1:500, SC-8008; Santa Cruz), rabbit anti-KRAS polyclonal antibody (1:1000, ab180772; Abcam), rabbit anti-GAPDH polyclonal antibody (1:2000, ab9485; Abcam), rabbit anti-phosphorylated ERK1/2 (p-ERK1/2) (1:2000, #4376; CST) and anti-total ERK1/2 (t-ERK1/2) monoclonal antibodies (1:2000, #4695; CST), rabbit anti-phosphorylated AKT1 (p-AKT1) (1:1000, #9018; CST) and anti-total-AKT1 (t-AKT1) monoclonal antibodies (1:1000, #2938; CST) and rabbit anti-histone H3 polyclonal antibody (1:3000, ab1791; Abcam), followed by secondary antibody with a dilution of 1:2000 (goat anti-rabbit antibody, ab7090, Abcam; goat anti-mouse antibody, ab97040, Abcam). The amount of each target gene was normalized to GAPDH or histone H3 in each sample. The blots were visualized using an enhanced chemiluminescence reagent (Thermo, USA).