Ecl plus developing system

This analysis was conducted as previously described.14 Cells were lysed in RIPA buffer to obtain total protein samples and the proteins were separated by SDS‐PAGE in a 10% gel and then electrophoretically transferred to a nitrocellulose membrane (Bio‐Rad, Hercules, CA). The membranes were blocked with 5% non‐fat milk and then incubated with the following primary antibodies: anti‐EYA4, anti‐c‐JUN, anti‐p‐c‐JUN(ser73), anti‐VEGFA and anti‐CD31 (Cell Signalling Technology, Beverly, MA) overnight at 4°C. GAPDH served as a loading control. The membranes were incubated with a horseradish peroxidase–conjugated secondary antibody. The protein bands were detected with the ECL Plus Developing System (Amersham Biosciences; Piscataway, NJ).

Cell lysates were made with radioimmunoprecipitation assay buffer (RIPA) and western blotting was performed. Membranes were probed with antibodies for CK2α 1:5000 (Cell Signaling, 2656), phosphorylated CK2 substrate 1:10,000 (Cell Signaling, 8738), PARP 1:5000 (Cell Signaling, 9542), β-catenin 1:10,000 (Cell Signaling, 9562), HRP conjugated β-Actin 1:50,000 (Cell Signaling, 5125), or TCF8 (Cell Signaling, 3396). Horseradish peroxidase-conjugated secondary antibodies (Jackson Labs) were incubated for 1 h at room temperature. Blot development was performed with ECL Plus developing system (Amersham Biosciences).