Rabbit anti human β catenin antibody

Whole cell extracts from HL60 cells were prepared, and protein concentration was quantified with the Bradford method. Samples containing 100 μg of protein were separated by 15% SDS-PAGE gel electrophoresis, and transferred to PVDF membrane. Membranes were blocked and incubated with primary antibodies, as indicated for each target protein, at 4°C overnight. Next, membranes were washed and incubated with horseradish peroxidase (HRP) labeled secondary antibodies, then developed with an ECL kit (Pierce, MA, USA), following the manufacturer's instructions. Quantity One 4.6.2 software was used for gray-value analysis of the electrophoresis bands in each group, to compare differential levels of ICAT, β-catenin and TCF-4 proteins and the Wnt signaling pathway downstream target proteins TCF-1, cyclin D1 and c-Jun. Rabbit anti-human β-catenin antibody was from Abcam (Cambridge, England), goat anti-human ICAT, α-tubulin, TCF4, cyclinD1, c-Jun and TCF1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

PLC5 cells were plated on glass coverslip and cultured overnight to 30% confluence. Cells were then treated with bufalin or vehicle for 24 hours followed by fixation with 3% paraformaldehyde and permeation with 0.1% Triton X-100. Nonspecific binding sites were blocked with 1% BSA for 30 minutes. The cells were incubated with primary mouse anti-human CCRK antibody (Sigma) or rabbit anti-human β-catenin antibody (Abcam) for 1 hour, and then incubated with FITC-conjugated goat-anti-mouse antibody (Invitrogen) or rhodamine-conjugated goat-anti-rabbit antibody (Invitrogen) for 30 minutes. Nuclei were counterstained by DAPI (Invitrogen) and Images were captured using confocal microscope (Olympus).