After transfection and curcumin treatment, cell protein was extracted and protein concentration was measured using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Protein lysates were loaded and separated on 10% SDS-polyacrylamide gels, then transferred 2 h in cold transfer buffer. Membranes were blocked in 5% fat-free milk for 2 h at room temperature and then incubated with primary antibodies and secondary antibodies as described previously.33 (link) Protein bands were visualized using enhanced chemiluminescence detection reagent (Thermo Fisher Scientific) and Image Lab™ software 4.1 (Bio-Rad Laboratories, CA, USA). The protein bands were analyzed as a percentage of β-actin levels.
For Co-IP assays, cells were cultured in 10 cm dishes (Corning) and divided into untreated (mock) and curcumin-treated groups for 48 h. The cells were lysed in RIPA lysis buffer (Beijing Solarbio Science & Technology) on ice for 30 min, and precleared with protein-G agarose beads (Sigma) for 4 h at 4°C. Lysates were then incubated overnight at 4°C with anit-Gli1 or anti-β-catenin primary antibodies, on a rotating wheel. The next day, antibody-antigen complexes were analyzed by Western blotting as described.
For Co-IP assays, cells were cultured in 10 cm dishes (Corning) and divided into untreated (mock) and curcumin-treated groups for 48 h. The cells were lysed in RIPA lysis buffer (Beijing Solarbio Science & Technology) on ice for 30 min, and precleared with protein-G agarose beads (Sigma) for 4 h at 4°C. Lysates were then incubated overnight at 4°C with anit-Gli1 or anti-β-catenin primary antibodies, on a rotating wheel. The next day, antibody-antigen complexes were analyzed by Western blotting as described.
