Sc 166025

Frozen samples were thawed in CelLytic MT Cell Lysis Solution (Sigma Aldrich, Munich, Germany) with the addition of protease inhibitors, Benzonase -50 U/ul (Merck; Millipore, Bedford, MA, USA) and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Protein concentrations of whole cell lysates were determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Equal amounts of total protein (30 µg) were mixed with sample buffer and dithiothreitol (DTT) and resolved by SDS-PAGE. After the completion of the electrophoresis, the samples were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon; Millipore, Bedford, MA, USA) and incubated in a 4% BSA solution in Tris-buffered saline (TBS) with the addition of 0.1% Tween-20. Later on, the membranes were incubated with mouse anti-human SOX18 antibody -1:100 (sc-166025; Santa Cruz Biotechnology Inc.) for a night at 4ºC. At the end the membranes were treated with the peroxidase-conjugated donkey anti-mouse secondary antibody -1:3,000 (715-035-150; Jacksons Immunoresearch, Suffolk, UK) for 1 h, rinsed, and incubated with the Immun-Star-HRP Chemiluminescent Substrate (Bio-Rad, Hercules, CA, USA). Protein quantifications were based on the total protein normalization.