The co-immunoprecipitation assay was performed using Huh7 cells transfected with GLI1 expressing plasmid (Huh7 GLI1 cells). Protein lysate was extracted in immunoprecipitation buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM PMSF, 1 mM sodium vanadate and 10 mM sodium fluoride). For Co-IP, 10 μl protein A/G-agarose beads pre-conjugated with the antibody against CtBP2 or SNAI1 and IgG were utilized to isolate the immunocomplexes. The antibody-conjugated beads were shaken with cell lysates at 4°C overnight. The next day, isolated beads were washed twice with PBS buffer and twice with RIPA buffer. The supernatant was examined by Western blotting.
