Sc 271786

Protein isolation and western blots were performed as described before [48 (link)]. For analysis of phosphorylated proteins, cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer (89900, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (cOmpleteTM Tablets, 11697498001, Roche, Basel, Swiss; Sodium orthovanadate, 13721-39-6, Fivephoton Biochemicals, San Diego, CA, USA). Antibodies against rabbit anti-GLI1 monoclonal antibody (C68H3, 1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-GLI-2 (Dilution, C-10: sc-271786, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit Anti-HSF1-phospho(S326) antibody (1:5000, EP1713Y, abcam, Cambridge, UK), rabbit Anti-HSF1 antibody (1:1000, 4356, Cell Signaling Technology, Inc., Danvers, MA, USA), Mouse anti-HSP70/HSPA1A antibody (1:1000, MAB1663, R&D Systems, Inc. Minneapolis, MN, USA), Rabbit anti-HSP90 Antibody (1:1000, #4874, Cell Signaling Technology, Inc., Danvers, MA, USA) or mouse anti-β-ACTIN (sc-47778, 1:5000, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies against HRP-linked anti-rabbit immunoglobulins (1:10,000, 7074) and anti-mouse IgG (1:10,000, NXA931) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and GE Healthcare (Cytiva, Marlborough, MA, USA), respectively.

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).