Rabbit anti gli1

Cells were lysed in a RIPA lysis and extraction buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitors (Thermo Fisher Scientific) on ice. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to nitrocellulose membranes (GE, 10600001). Membranes were probed with primary antibodies against mouse anti-ERK (Cell Signaling, 9107), rabbit anti-phospho-Erk (Cell Signaling, 9101), rabbit anti-IFT88 (Proteintech, 13967-1-AP), mouse anti-ARL6 (Proteintech, 12676-1-AP), rabbit anti-GLI-1 (Cell Signaling, 2553), rabbit anti-AURKA (Cell Signaling, 4718), and mouse anti-β-actin (Sigma, A5441). Immunoreactivity bands were detected using a Pierce electrochemiluminescence western blotting substrate (Thermo Fisher Scientific).

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).

Immunoblots experiments were performed using the following primary antibodies: anti-DYNC1I2 (Atlas Antibodies, Bromma, Sweden, #HPA040619, 1:1000), mouse anti-β-Actin (C4) (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-47778, 1:1000) or mouse anti-β-Actin (Cell Signaling Technology, Danvers, MA, #3700, 1:1000), mouse anti-HHIP (Abnova Germany; #H00064399-M01; 1:1000); mouse anti-GLI2 (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-271786, 1:500); rabbit anti-GLI1 (Cell Signaling Technology, Danvers, MA, #V812,1:1000).