WNT5A Signaling in Breast Cancer

WNT5A-expressing breast cancer cells or parental breast cancer cells treated with PFKP siRNA, rWNT5A, XAV939, U0126 or not were washed with ice-cold PBS and lysed in ice-cold phosphorylation lysis buffer (PLB). The protein estimation, SDS-PAGE and visualization procedures were performed as described in Prasad et al. [40 (link)]. The following primary antibodies were used: anti-WNT5A from R&D systems (MN, USA); anti-Hexokinase-II, anti-Puruvate Kinase; anti-PKFP, anti-non-phospho (Active) β-catenin, and anti-pERK1/2 antibodies from Cell Signaling Technology (MA, USA); anti-MCT1 antibody from Santa Cruz Biotechnology Inc. (TX, USA); and anti-β-actin antibody from Sigma-Aldrich (MO, USA). The secondary antibodies used were goat anti-mouse, goat anti-rabbit and rabbit anti-goat, which were procured from Dako (Glostrup, Denmark). Separated protein bands were visualized using Chemiluminescence HRP substrate (Millipore), and the membranes were imaged and analyzed using the Chemi Doc™ imaging system from Bio-Rad.