FIG. 6.

Activation of TCF and LEF transcriptional activity by AML fusion proteins. (a) Analysis of LEF-1-bound catenin levels in the presence of AML fusion proteins. HA-LEF-1 was transfected along with the indicated fusion protein or a control vector into U937 cells. Equal protein concentrations of the transfected cell populations were used to immunoprecipitate HA-LEF-1 by an anti-HA affinity matrix. Significantly increased levels of LEF-1-bound plakoglobin were detected in Western blot analyses of the immunoprecipitates. Subsequent Western blot analysis of HA-LEF-1 served as control for transfection and equal loading. (b) Following transfection of HA-LEF1, PML-RARα, and AML1-ETO, a dose-dependent increase in LEF-1-bound plakoglobin and β-catenin was demonstrated by anti-HA immunoprecipitation and Western blotting. The immunoglobulin heavy chain and actin served as loading controls. (c) Chromatin immunoprecipitation analysis of the c-myc promoter. U937 cells with or without AML1-ETO induction were formaldehyde cross-linked, and DNA-protein complexes were precipitated with either antiplakoglobin or anti-β-catenin antibodies or a control antibody. Samples were washed and cross-links were reversed before promoter sequences were amplified by PCR.