Mouse anti fg

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).