Polyclonal rabbit anti e cadherin

Whole cell extracts were obtained by lysing cells in TNTE buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 0.5% Triton X-100, 1 mM sodium vanadate, and 25 mM sodium fluoride) containing protease inhibitors (5 μg/ml PMSF, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, and 0.5 μg/ml aprotinin). The detailed western blotting procedures have been described previously [25 (link)]. The protein samples were analyzed using monoclonal mouse anti-ARHGDIA (Santa Cruz Biotechnology, 1:5000), polyclonal rabbit anti-PCBP2 (Beijing Aviva Systems Biology, 1:2000), polyclonal rabbit anti-E-cadherin (Cell Signaling, 1:1000), polyclonal mouse anti-N-cadherin (BD Biosciences, 1:1000), polyclonal mouse anti-vimentin (Santa Cruz Biotechnology, 1:2000), polyclonal rabbit anti-Snail1 (Santa Cruz Biotechnology, 1:2000), polyclonal rabbit anti-Twist1 (Santa Cruz Biotechnology, 1:2000), mouse anti-β-actin (Sigma-Aldrich,1:5000) antibodies. The Quantity One software (Bio-Rad Laboratories) was used to quantify the relative expression levels of the target proteins.

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.