Beta-Catenin Activation Kit

The kit contains a polyclonal anti-beta-catenin rabbit primary antibody, a goat anti-rabbit secondary antibody conjugated to the DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence labeling of beta-catenin for high-content screening (HCS) assays.

Signaling through the Wnt pathway via the nuclear translocation of the key transcription factor beta-catenin is critical for cell growth and differentiation during development. Beta-catenin is located mostly in the adherens junctions of epithelial cells associated with E-cadherin, but also in cytoplasmic pools complexed with APC. In the absence of Wnt signaling, beta-catenin is phosphorylated by both casein kinase 1α (CK1α) and glycogen synthase kinase 3ß (GSK3ß), thus targeting this protein for ubiquitination and subsequent proteolysis. Activation of beta-catenin with Wnt ligands or through kinase inhibition results in its dissociation with the APC and cadherin complexes, and its translocation to the nucleus. Once in the nucleus, beta-catenin binds with other transcription factors, such as LEF/TCF, to activate transcription of target genes. Constitutive pathway activation with or without ligand or beta-catenin stabilization results in proliferation and cancer, prevalently in colon, breast and prostate cancers.

Beta-catenin translocation in HT1080 cells treated GSK-3 inhibitor X was quantitatively assayed using the Beta-Catenin Activation Kit, the ArrayScan® HCS Reader and the Compartmental Analysis BioApplication Software Module. Phosphorylation inhibition by GSK-3 Inhibitor X results in beta-catenin stabilization and its subsequent translocation to the nucleus. Therefore, the assay output parameter is the measurement of nuclear intensity or the difference between the cytoplasm and nuclear intensity after treatment. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

Figure 2. Beta-catenin translocation in HT1080 colon fibrosarcoma and HeLa cervical carcinoma cells treated with GSK-3 Inhibitor X. HT1080 cells were treated with 10 µM (20 hours) to measure nuclear translocation of beta-catenin. Cells were then fixed and stained with anti-beta-catenin primary antibody and DyLight 549 Goat Anti-Rabbit secondary antibody. Cells were also labeled with Hoechst 33342 dye for nuclear staining. Top panels are the non-treated cells; bottom panels are the treated cells. HeLa Cells were treated for twenty hours with the indicated drug concentrations. Beta-catenin moves from the membrane periphery to the nucleus with increasing concentrations of drug. 

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