Protein Expression Profiling of Colon Cancer Cell Lines

HCT8, SW480 cell lines and their N-cadherin plasmid treated variants as well as derived colon cancer primocultures 47B and 48B were grown in 6-well plates (150,000 cells/mL). Then, the cells were washed with PBS and harvested in ice-cold lysis buffer. Tumors excised from mice (both variants of HCT8 tumors and 47B, 48B tumors) were homogenized using Tissue lyzer (2 cycles—25 vibration/s; 4 °C; 1 min; Qiagen, Germantown, MD, USA) in ice-cold lysis buffer. Protein content of resulting cell lysates was determined by BCA assay. Detailed description of SDS-PAGE of diluted cell lysates (30 µg/well), protein transfer to PVDF membrane, antibody incubation and protein detection is summarized in [26 (link)]. The following primary antibodies from Cell signaling technology were used: polyclonal rabbit anti-E-cadherin 1:2000; polyclonal rabbit anti-N-cadherin 1:1500; polyclonal rabbit anti-vimentin, 1: 2000; monoclonal rabbit anti-CD44, 1:2000. Monoclonal rabbit anti-MRP1, 1:2500; monoclonal rabbit anti-MDR1, 1:2500, monoclonal rabbit anti-FAK, 1:5000; monoclonal rabbit anti-GEF-H1, 1:2000 and housekeeping monoclonal mouse α-tubulin, 1:10,000. Housekeeping monoclonal mouse β-actin, 1:10,000 was from Sigma Aldrich. Samples were analyzed by Azure Biosystems c600 and semi-quantitative analysis was performed using program Azure spot.