Western Blot Analysis of Extracellular Matrix Proteins

After treatment, the cells were washed three times with pre-cooled PBS, and the total protein of cell lysates was extracted with RIPA buffer (Santa Cruz Biotech.). The specific protein was detected by Western blot analysis as previously described[13 (link)]. In brief, the cell lysates or S3 pellet in media were boiled and then electrophoresed in 12% SDS-PAGE acrylamide gels. They were then transferred onto nitrocellulose membranes (Bio-Rad) and incubated for 1 h in TBS-T containing 5% nonfat milk. The membranes were incubated overnight at 4°C with primary antibodies against fibronectin (Santa Cruz Biotech.), laminin β-1 (Santa Cruz Biotech.), Cav-1 (Santa Cruz Biotech.), and ERK phosphorylation (Cell Signaling). The membranes were washed three times in TBS-T and incubated for 1 h at room temperature with corresponding HRP-conjugated anti-rabbit or anti-mouse antibodies (Santa Cruz Biotech.). The membranes were developed with the SuperSignal West Pico HRP substrate kit (Pierce) and photographed on a Kodak 4000 image station (Carestream Molecular Imaging). To control sample loading and protein transfer, we stripped and reprobed the membranes with β-actin (Santa Cruz Biotech.) or total ERK (Cell Signaling) antibody.