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Materials and methods
Yeast two-hybrid screen and X-gal assay. A yeast two-hybrid array screen using the LexA-TCF4 as bait was performed as described previously [18]. The X-gal activities were determined from three separate liquid yeast cultures according to the instructions of the Galacto-light Plus kit (Tropix Inc., Bedford, MA).
Immunoprecipitation, Western, and GST-pull-down analyses. Cells transfected with or without indicated constructs were lysed directly in AEBSF modified RIPA buffer and further subjected to immunoprecipitation and Western analyses as described previously [14] and [16]. Anti-HA antibody was purchased from Covance, anti-Gal4, anti-cyclin D1, and anti-c-Myc from Santa Cruz, anti-β-catenin from BD Biosciences, anti-Daxx, and anti-β-actin from Sigma and anti-TCF4 from Upstate biotechnology. GST pull-down assay were performed as described [14] and [16] using 3 μg recombinant GST-Daxx570–740 fusion proteins along with indicated in vitro-synthesized 35S-labeled proteins. After binding and washing, bound proteins were fractionated by SDS–PAGE and analyzed by autoradiography.