Sdc-1 Regulation of DLL4 Expression

Briefly, total cell lysates of control and Sdc-1 siRNA transfected SUM-149 cells were prepared as described before [3 (link)]. Protein concentration was determined using Bradford assay and 50 ug protein per lane were electrophoresed on 10% SDS-PAGE and electrotransferred into nitrocellulose membrane (Millipore, Germany). After blocking with 5% BSA in tris- buffered saline with 0.1% tween (TBST) for 1 hour the membrane was probed with 2 ug/mL primary antibody against DLL4 (Santa Cruz Biotech, CA, USA) overnight at 4 °C. On the next day, the membrane was incubated with 1 ug/mL anti-mouse secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotech, CA, USA) for 1 hour at room temperature. The immunoreactivity was visualized by Enhanced Chemiluminescence (ECL) reaction and BioSpectrum 815 Imaging System (Analytik Jena, USA). β-actin (Santa Cruz Biotech) was used as a loading control.