Superwest signal pico

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).

Total proteins were extracted by sonication in radioimmunoprecipitation assay RIPA buffer) containing protease and phosphatase inhibitors (Complete Mini Protease Inhibitor Cocktail Tablets and PhosSTOP Inhibitor Tablets for phosphatases, both Roche, Basel, Switzerland). Protein concentration was measured using the BCA (Bicinchoninic Acid) kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (50 μg) were separated on 7% or 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham BioSciences, Little Chalfont, England, UK). The quality of transfer was determined with Naphthol Blue Black (Sigma-Aldrich, St. Louis, MI, USA, SAD) staining. Membranes were blocked with 5% milk-TBST (Tris-Buffered Saline, 0.1% Tween^® 20 Detergent) solution and incubated with primary antibodies overnight. Primary antibodies used in this study are listed in Supplementary Table S2. After overnight incubation, membranes were washed in TBST and incubated for 1 h with the appropriate secondary HRP-conjugated antibodies—anti-rabbit 1:6000 (554021, BD Pharmingen, San Jose, CA, USA) and anti-mouse 1:8000 (554002, BD Pharmingen). Proteins were visualized using SuperWest Signal Pico and Femto reagents (Thermo Fisher Scientific, Waltham, MA, USA) on the Uvitec Image Alliance Q9 mini instrument (Uvitec, Cambridge, England, UK).