Anti β catenin monoclonal antibody

Total cell lysates were extracted with 50 mM Tris pH7.5, 0.1% IGEPAL, 100 mM NaCl, 1 mM MgCl₂, 5 mM EDTA and protease inhibitors and sonicated. 25 µg of lysate from each cell line was separated on a 10% SDS-PAGE gel and transferred to Immobilon P membrane (Millipore). Blots were probed with 1:1000 anti-β-catenin monoclonal antibody (BD Biosciences), 1:1000 anti-active (unphosphorylated) β-catenin (EMD Millipore) and 1:1000 anti-actin monoclonal antibody (Sigma) in blocking buffer (5% nonfat dried milk in TBST).

Proteins were fractionated on an SDS-PAGE gel and transferred to an Immobilon-P PVDF membrane (0.45 μm, Millipore). After blocking, membranes were incubated with primary antibodies and then anti-rabbit or anti-mouse secondary antibodies were used for detection. Immunoreactive proteins were visualized using Western Lightening Plus-ECL (PerkinElmer, #NEL105001EA). The following primary antibodies were used: anti-β-actin monoclonal antibody (Sigma, #A5441); anti-USP5 monoclonal antibody (Santa Cruz, #sc-390,943), anti-β-catenin monoclonal antibody (BD Biosciences, #610,153), anti-Slug polyclonal antibody (Santa Cruz, #sc-10,436), anti-E-cadherin monoclonal antibody (BD Biosciences, #610,182), anti-N-cadherin monoclonal antibody (BD Biosciences, #610,921), anti-Vimentin monoclonal antibody (Cell Signaling Technology, #5741S), and anti-Ubiquitin monoclonal antibody (Santa Cruz, #sc-8017).

For detection of tyrosine phosphorylation, the proteins were resolved on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with anti-phosphotyrosine primary antibody (Millipore, 4G10 Platinum-HRP), followed by IRDye 800CW Rabbit anti-HRP secondary antibody (LI-COR). The blots were imaged and quantitated using an Odyssey Infrared Imaging System (LI-COR) and Image Studio software.
Expression of receptor constructs in cells used for the luciferase assay was determined by immunoblot detection of receptor proteins using anti-Flag M2 antibody (Sigma) and 10 µl of the lysate from the luciferase assays. Cytosolic β-catenin, in cells transfected with empty vector, ROR1, ROR2 or MuSK constructs and treated with Wnt3a and/or Wnt5a was detected using the anti-β-catenin monoclonal antibody (BD Biosciences). For this assay, cells were lysed in a hypotonic buffer (20 mM Tris HCl pH 8.0, 1 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 1 mM DTT with protease inhibitors) and the cytosolic fraction used for electrophoresis. The proteins were electrophoresed on 4–20% TGX gels (Bio-Rad), followed by transfer to PVDF membrane. Blots were incubated with the primary antibodies (overnight), and anti-mouse or rabbit IR conjugated secondary antibodies (1 hour, Invitrogen) and the proteins visualized and quantitated using the Odyssey Infrared Imaging System (LI-COR).