Mouse anti actin

All cell lines used in this study were purchased from ATCC and cultured as following: A549 cells with F‐12K medium (Gibco) containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Sangon Biotech), H1299, H1975 and H1650 with RPMI‐1640 medium (Gibco) containing 10% FBS and 1% penicillin/streptomycin, 16HBE cells with DMEM (Gibco) medium containing 10% FBS and 1% penicillin/streptomycin. Cell transfection with plasmids or siRNAs was carried out as previously described.¹⁷ The antibodies used for Western blot were as follows: mouse anti‐CRKL (1:1000; Santa Cruz); mouse anti‐Flag tag (abbreviation for Fg; 1:5000; Sigma); rabbit anti‐GLI2 (1:1000; ABclonal); and mouse anti‐ACTIN (1:5000; Genscript). The siRNAs sequences were shown as follows: MOCK‐siRNA, 5′‐CAA ACA CUU CCU UGG AAU GdTdT‐3′; CRKL‐siRNA‐1, 5′‐GCU CUG CUC UAC CAU GUU UdTdT‐3′; CRKL‐siRNA‐2, 5′‐CGT GAA AGU CAC AAG GAU GdTdT‐3′; GLI2‐siRNA‐1, 5′‐GUU CCU CAC GGC GUC GUA GdTdT‐3′; GLI2‐siRNA‐2, 5′‐CAA GAC CGA GCC UGA GGG CdTdT‐3′. For chemical treatment, cells were seeded into 96‐well plate at 1 × 10⁴/well. After 24 hours, cells were treated with 5 μg/mL adriamycin (Sigma), 20 μg/mL 5‐FU (Sigma), 10 μg/mL cisplatin (Sigma), 20 μmol/L cyclopamine (MedChemExpress), 10 μmol/L gefitinib (MedChemExpress) or 10 μmol/L GANT61 (MedChemExpress) for additional 24 hours.

Cells were transfected using lipofectamine 2000 according to the manufacturer's instructions. Forty‐eight hours after transfection, cells were harvested for immunoprecipitation and western blot analysis with standard protocols. To examine the ubiquitination levels of GLI2 and GLI3, A549 cells were transfected with Myc‐GLI2, HA‐GLI3 and different SPOP mutants. Before cell harvesting, the cells were treated by MG132 (50 mmol/L/mL) for 4 hours to prevent protein destabilization. Cells were first lysed by 100 mL denaturing buffer (1% SDS, 50 mM Tris, pH 7.5, 0.5 mmol/L EDTA and 1 mmol/L DTT) and incubated at 100°C for 5 minutes. The lysates were diluted with 900 mL lysis buffer and subjected to immunoprecipitation and western blot. The antibodies used for western blot analyses were as follows: mouse anti‐Fg (Sigma, Darmstadt, Germany); mouse anti‐ACTIN (Genscript, Corporation, Piscataway, NJ, USA); rabbit anti‐GLI1 (ABclonal, Woburn, MA, USA); rabbit anti‐PTCH1 (ABclonal); rabbit anti‐BCL2 (ABclonal); rabbit anti‐HHIP (ABclonal); rabbit anti‐AXIN2 (ABclonal); rabbit anti‐c‐Myc (ABclonal); rabbit anti‐CTGF (ABclonal); rabbit anti‐AREG (ABclonal); rabbit anti‐SPOP (ABclonal); mouse anti‐Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti‐HA (Santa Cruz); mouse anti‐Ub (Santa Cruz); goat antimouse HRP (Abmax) and goat anti‐rabbit HRP (Abmax, Beijing, China).