Image alliance 4.7 instrument

Whole-cell protein extraction, determining the protein concentration, and western blot technique were performed as previously described [20 (link)]. The membranes were probed with following primary antibodies: rabbit anti-GLI1 1:300 (V812, Cell Signaling Technology, Danvers, MA, USA), mouse anti-GLI2 1:100 (sc-271786, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-GLI3 1:1000 (GTX104362, GeneTex, Irvine, CA, USA), rabbit anti-PTCH1 1:1000 (17520-1-AP, ProteinTech, Rosemont, IL, USA) and mouse anti-β-actin 1:4000 (60008-1-Ig, ProteinTech, Rosemont, IL, USA) was used as loading control. After overnight incubation, membranes were washed in TBST (Tris-Buffered Saline, 0.1% Tween^® 20 Detergent) and incubated for 1 h with appropriate secondary HRP-conjugated antibodies, anti-rabbit 1:6000 (554021, BD Pharmingen, San Jose, CA, USA) and anti-mouse 1:8000 (554002, BD Pharmingen, San Jose, CA, USA). Proteins were visualized using SuperWest Signal Pico and Femto reagents (Thermo Fisher Scientific, Waltham, MA, USA) on Uvitec Image Alliance 4.7 instrument (UVItec, Cambridge, England, UK).

Total proteins were extracted by sonication in RIPA (Radioimmunoprecipitation assay buffer) buffer containing protease and phosphatase inhibitors, and the protein concentration was measured using the BCA (Bicinchonic Acid) kit (Thermo Fisher Scientific, Waltham, MA, USA); 40 μg of protein was loaded on 7% PAA (Polyacrylamide) gel. After electrophoresis, they were transferred to a nitrocellulose membrane (Amersham BioSciences, Little Chalfont, England, UK), blocked with 5% milk and incubated with primary antibodies overnight. Antibodies used for detection were as follows: rabbit anti-GLI1 (Cell Signaling Technology, V812, 1:200, Danvers, MA, USA), mouse anti-GLI2 (Santa Cruz Biotechnology, sc-271786, 1:200, Dallas, TX, USA), rabbit anti-GLI3 (GeneTex GTX104362, 1:1000, Irvine, CA, USA). Actin (60008-1-Ig, ProteinTech, 1:4000, Rosemont, IL, USA) was used as a loading control. After washing in TBST (Tris-Buffered Saline, 0.1% Tween^® 20 Detergent), secondary antibodies HRP (Horseradish Peroxidase)-conjugated anti-rabbit (BD Pharmingen, 554021, 1:6000, San Jose, CA, USA) and anti-mouse (BD Pharmingen, 554002, 1:8000, San Jose, CA, USA) were applied for 1h at room temperature, washed, and visualized using SuperWest Signal Pico and Femto reagents (Thermo Fisher Scientific, Waltham, MA, USA) on Uvitec Image Alliance 4.7 instrument (UVItec, Cambridge, England, UK).