Western Blot Analysis of Smad, BCAR3 and p130Cas Proteins

Cells were lysed with RIPA buffer containing 1% Triton X-100, protease inhibitors and phosphatase inhibitors. Total protein lysates were quantified, and lysates containing 50 μg of total protein were separated by SDS-PAGE and then transferred onto nitrocellulose membranes and subjected to Western blot analysis as previously described [28 (link)]. Densitometry of Western blots was quantified using Quantity One 1-D analysis software (Bio-Rad Laboratories, Hercules, CA, USA).
To obtain nuclear extracts, cells were lysed with phosphate-buffered saline (PBS) containing 1% Nonidet P-40. The nucleus were washed in the lysis buffer multiple times and lysed with loading dye containing SDS, as described in a protocol developed by others [29 (link)].
The primary antibodies used for Western blot analysis were rabbit polyclonal Smad2/3 antibody (sc-8332; Santa Cruz Biotechnology), rabbit phospho-Smad3 antibody (9520; Cell Signaling Technology, Danvers, MA, USA), goat polyclonal BCAR3 antibody (sc-47811; Santa Cruz Biotechnology), rabbit polyclonal p130Cas antibody (sc-860; Santa Cruz Biotechnology), rabbit polyclonal USF-2 antibody (sc-862; Santa Cruz Biotechnology) and mouse monoclonal β-tubulin antibody (sc-5274; Santa Cruz Biotechnology). All corresponding secondary antibodies were purchased from Santa Cruz Biotechnology.