Anti smad5

The JB6 cell line was used. At 24 h after seeding, PNU-74654 (Selleck, China), LiCl (Sigma, USA) or DMSO (Sigma, USA) was added into the culture medium. At 48 h after seeding, the cell pellets were collected following treatment with 0.25% trypsin. The total protein from the cell pellets were extracted respectively. SDS-PAGE was performed with 0.8% polyacrylamide gels. Proteins were then transferred to PVDF membranes (Millipore, USA), and membranes were blocked with 5% nonfat dry milk, in TBST (Tris-buffered saline containing 0.1% Tween-20). The PVDF membranes were then incubated with diluted primary antibodies overnight at 4 °C, washed with TBST, incubated with the HRP-labeled secondary antibodies (Sangon Biotech, China), and washed with TBST again. Finally, ECL (Bio-Rad, USA) was added to the PVDF membrane. The results were observed and recorded in a gel imager (Bio-Rad, USA). Primary antibodies against the following proteins were used: CyclinD1 (1:500, Sangon Biotech, China), β-Catenin (1:1000, Santa Cruz, USA), p-β-Catenin (1:5000, CST, USA), LEF1 (1:1000, Santa Cruz, USA). Anti-Smad 5 (1:1000, proteintech, China) was used to detect the expression of Smad5 protein after treatments.