Tcf 1

Co-immunoprecipitation experiments were performed in whole cell extracts of CuB-treated A549 and H1299 cells. For immunoprecipitation analysis, 50 μL of Sepharose A beads (Sigma-Aldrich) were incubated with 2.5 μg of β-catenin (Invitrogen: 71-2700) antibody diluted up to 500 μL in wash buffer containing PBS with 2% triton-X-100 overnight at 4 °C. After centrifugation and washing, these antibody-coated beads were incubated with 500 μg of whole cell extracts from each sample for 1 h at 4 °C. After, centrifugation, the supernatant was collected. β-actin was used as an equal loading control. Beads were washed thrice and equal volumes of protein from each sample were mixed with laemmlli buffer, boiled in a water bath and loaded onto 12% SDS-PAGE. Immunoblotting analysis was performed by using TCF-1 (Life Technologies: A13969) antibody. The blots were incubated with specific HRP-conjugated secondary anti-rabbit antibody and bands were visualized using ECL (Millipore) on ImageQuant LAS4010 chemiluminescence detection system (GE Healthcare, Amarsham, UK).

Whole cell lysates of A549 and H1299 cells at 24 h of CuB treatment were prepared using the RIPA-lysis buffer (Millipore, Billerica, MA). Protein extraction from tumor samples was also performed using RIPA-lysis buffer. Proteins were resolved on 10–12% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). After incubation in blocking buffer (5% skimmed milk or 2% BSA in TBST) for 1 h, the membranes were incubated with primary antibodies specific for Wnt3/3a (Abcam: ab172612), Fzd-7 (Sigma-aldrich: AV41251), phospho-GSK-3α (Cell Signaling Technology: 9337), GSK-3α (Abcam: ab40870), phospho-GSK-3β (Santa Cruz: SC-11757), GSK-3β (Abcam: ab18893), β-catenin (Invitrogen: 71-2700), TCF-1 (Life Technologies: A13969), MMP-2 (Cell Signaling Technology: 4022S), E-cadherin (Santa cruz: SC-8426), COX-2 (Millipore: AB5118), MYC (Millipore: 06-340), Cyclin D1 (Santa cruz: SC-8396), Survivin (Cell Signaling Technology: 2808S), VEGF (Santa cruz: S C-53462), Vimentin (Santa cruz: SC-6260), PCNA (Santa cruz: SC-7907) and β-actin (Cell Signaling Technology: 4970L). The blots were then incubated with specific HRP-conjugated secondary antibodies and bands were visualized using ECL (Millipore) on ImageQuant LAS4010 chemiluminescence detection system (GE Healthcare, Amarsham, UK). The band intensities were quantified using ImageJ 1.46r software.