Western Blotting Protocol for Protein Expression Analysis

Western blotting was performed using established methodology [13 (link)]. Briefly, cells were washed with PBS and lysed in RIPA buffer. Proteins (20–30 μg) were separated by 10–12% SDS/PAGE and then transferred on an immunoblot PVDF membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% nonfat milk, the membrane was incubated with primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Technology, sc-2033 for anti-goat, and sc-2004 for anti-rabbit, 1:3000) and then developed using ECL solution (Pierce). Primary antibodies used were rabbit anti-β-catenin (Abcam, ab32572, 1:1000), rabbit anti-fibronectin (Santa Cruz Technology, sc-9068, 1:1000), rabbit anti-Wnt3 (Abcam, ab32249, 1:1000), rabbit anti-Wnt7B (Abcam, ab94915, 1:1000), and goat anti-actin (Santa Cruz Technology, sc-1616, 1:3000). For protein expression quantitation, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image/).