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. 2013 Nov 15;27(22):2473–2488. doi: 10.1101/gad.229062.113

Figure 4.

Figure 4.

β-Catenin recruits the α-catenin:APC complex to LEF-1/TCF complexes. (A) MudPIT analysis of affinity-purified Flag:β-catenin and Flag:LEF-1 complexes isolated from HEK293 cells without and with 100 nM Factor XV, respectively. Shown is a subset of the identified interacting proteins ranked in abundance according to the total number of peptides identified by mass spectrometry and the percentile of protein sequence coverage. (B) SDS-PAGE and silver stain analysis of Flag:β-catenin, Flag:LEF-1, and Flag:TCF4 complexes isolated from untreated and 100 nM Factor XV-induced HEK293 cells, as indicated. (C) Immunoblot/co-IP analysis of Flag:LEF-1 complexes affinity-purified from HEK293 cells transfected with control-specific (si CTL), α-catenin-specific, or β-catenin-specific siRNAs. (Bottom panels) The knockdown efficiency of each siRNA was analyzed by immunoblot of the lysate. Where indicated, cells were treated for 4 h with 100 nM Factor XV. (D) Immunoblot time-course analysis of native LEF-1 immunoprecipitates in Wnt3a signaling HEK293 cells. Cells were incubated with Wnt3a-conditioned medium for the times indicated. Immunoblot analysis of the input nuclear fraction is shown in the bottom panels. (E) ChIP analysis of the AXIN2, DKK1, or SP5 genes in HEK293 cells treated with Wnt3a-conditioned medium for the indicated times. Factor occupancy at the WRE (black) or coding (gray) regions was measured using the specific antisera indicated above each panel. Bars represent the average of triplicate experiments.