Western Blot Analysis of Hedgehog Pathway

Cells were lysed in cold RIPA buffer (50mM Tris-HCl pH 7.5, 1% NP-40, 150mM NaCl, 5mM EDTA, 0.25% NaDOC, and 0.1% SDS) supplemented with protease and phosphatase inhibitors and centrifuged at 20,000× g for 20 min at 4°C [26 (link)]. Supernatant was collected as whole cell extract (WCE). Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and incubated for 1 h in blocking buffer at room temperature. The following primary antibodies were used: mouse anti-GLI1 (#2643, Cell Signaling Technologies, Danver, MA, USA), mouse anti-SMO (sc-166685, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse anti-HSP90α/β (sc-13119, Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with the corresponding horseradish peroxidase (HRP)-coupled secondary antibody, membranes were developed by using SuperSignal West Femto (Thermo Fisher Scientific, Waltham, MA, USA) and imaged with ChemiDoc Imaging Systems (Bio-Rad, Hercules, CA, USA).