Figure 4

Wnt3a stimulation of 293T cells promotes the convergence of β-catenin and Tiam1/Rac1 pathways. 293T cells were treated with L-CM (L) or Wnt3a-CM (W) for 18 hours. (A) Activated Rac1-GTP level was determined by GST pull-down assay using the PAK binding domain (PBD), followed by Western blotting analysis using Rac1-specific antibody. Total Rac1 expression was determined by Western blotting analysis of whole cell lysates. (B) Cell extracts were immunoprecipitated (IP) with either Tiam1 antibody (top) or β-catenin and TCF-4 antibodies (bottom) and analyzed by Western blotting using indicated antibodies. Whole cell lysates (WCL) were analyzed by immunoblotting (IB) to confirm expression of endogenous proteins as shown. Blots were probed with β-actin antibody as a loading control. (C) Cytoplasmic (CE) and nuclear extracts (NE) were analyzed by Western blotting using Tiam1 and β-catenin antibodies. Topoisomerase II and Paxillin antibodies were used to confirm the purity of nuclear and cytoplasmic fractions, respectively. (D) For ChIP assay, chromatin was subjected to immunoprecipitation using TCF-4, β-catenin, Rac1, and Tiam1 antibodies. Enriched chromatin was analyzed by PCR using primers that flank the TBEs in Wnt target genes, c-Myc and Cyclin D1. GAPDH primers, which flank a region of the GAPDH promoter that is devoid of TBEs, were used as a negative control.