Protein Extraction and Western Blotting Analysis

Total proteins were extracted with protein extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) from melanoma cell lines M21 and A375 and from their derivative cell lines produced by the above-mentioned plasmid transfection or viral infection. Nuclear and cytoplasmic proteins were extracted using nuclear and cytoplasmic extraction reagents, respectively (Pierce Biotechnology, Rockford, Illinois, USA). Western blotting assays were used to determine the protein levels of hSulf-1 (rabbit anti-human sulfatase 1, Abcam Company Ltd., Shanghai, China), total AKT (t-AKT), phosphorylated AKT (p-AKT, mouse anti-AKT and rabbit anti-phosphorylated AKT, Cell Signaling Technology Inc., Shanghai, China) and CDK4 (mouse anti-CDK4, Cell Signaling Technology) [23 (link)].