2.4. GLI1 Western blot analysis

The following procedures were carried out at 4°C. Approximately 1 × 10⁶ cells were resuspended and incubated in 0.15 mL of RIPA buffer (Sigma) and protease inhibitor cocktail (Pierce) for 5 minutes with rocking. The protein concentration was determined with BCA assay kit (Pierce). All the following procedures were carried out at room temperature. The GLI1 protein was separated on SDS‐PAGE gels, transferred onto a nitrocellulose membrane, and incubated in PBST buffer with 5% milk for 30 minutes. The membrane was washed with PBST buffer, incubated with polyclonal rabbit GLI1 antibody (Cell Signaling Technology; cat. no. 2354) (1:15 000 dilution) in PBST buffer with 5% milk for overnight at 4°C. The membranes were washed with PBST buffer (1X PBS, 0.3% Tween‐20) and incubated with secondary antibody conjugated with HRP (Donkey anti Rabbit IgG‐HRP, Santa Cruz Biotech; cat. no. sc‐2077) for 1 hour (1:15 000 dilution in PBST with 5% milk). The membrane was then washed 3× with PBST buffer. The GLI1 protein was visualized using SuperSignal West Femto chemiluminescence kit (Thermo Fisher Scientific). For GAPDH Western blot analysis, the same membrane was stripped with stripping buffer (Thermo Fisher) for 30 minutes at room temperature and then incubated in PBST buffer with 5% milk for 30 minutes. The membrane was washed with PBST buffer and incubated with polyclonal rabbit GAPDH antibody (Cell Signaling Technology; cat. no.14C10) (1:30 000 dilution) in PBST buffer with 5% milk for 1 hour at room temperature. The membranes were then washed with PBST buffer and incubated with secondary antibody conjugated with HRP (Donkey anti Rabbit IgG‐HRP, Santa Cruz Biotech; cat. no. sc‐2077) for 1 hour (1:30 000 dilution in PBST with 5% milk) at room temperature. The membrane was then washed 3× with PBST buffer. The GAPDH protein was visualized using SuperSignal West Pico chemiluminescence kit (Thermo Fisher Scientific).