Western Blot Analysis of Hedgehog Signaling Proteins

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).