Ripa lysis buffer

Western blot analysis was performed as described previously (11 (link)). Total proteins extracted from frozen tissues and cells lines transfected for 72 h and then incubated with RIPA lysis buffer (DBI Bioscience, Shanghai, China), following the manufacturer's protocol. After protein quantitation using a BCA Protein Quantitative kit (DBI Bioscience), 100 µg protein per lane were separated by SDS-PAGE (10% gel) and blotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then incubated overnight at 4°C with anti-NEDD9 (1:1,000; cat. no. ab37161; Abcam) or anti-tubulin (cat. no. sc-8035; 1:1,000; Santa Cruz Biotechnologies, Inc., Dallas, TX, USA), followed by incubation with horseradish peroxidase-conjugated IgG goat anti-rabbit H&L; (cat. no. ab97051; 1:2,000; Abcam) and goat anti-mouse IgG H&L (cat. no. ab6708; 1:2,000; Abcam) at room temperature for 1 h. An enhanced chemiluminescence kit (Merck KGaA) was used for detection using FluorChem™ Q software (version 3.4.0.0; Protein Simple, San Jose, CA, USA). The experiments were performed in triplicate.

Total cellular protein was extracted from cell lines transfected for 72 h using RIPA lysis buffer (DBI Bioscience, Shanghai, China) following the manufacturer’s instructions. After protein quantitation using BCA Protein Quantitative Kit (DBI Bioscience), equal amounts of proteins were separated by SDS-PAGE and blotted onto to PVDF membranes (0.45 µm; Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk and then incubated overnight at 4°C with anti-NEDD9 (1:1,000; Abcam) or anti-tubulin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), followed by incubation with horseradish peroxidase-conjugated IgG. An ECL kit (Millipore) was used for detection.