Mutants of BCL9 proteins that cannot bind to β-catenin. A, Sequence alignments of the HD2 domains of Lgs, BCL9 and BCL9-2/B9L (residue numbers in the latter two refer to mouse or human HD2 sequences, which are identical). Individual conserved residues (boxed) were mutated, and semiquantitative estimates of their in vitro binding affinities (++/+/-) to the β-catenin ARD are given underneath. B, pull-down assays between in vitro translated wt and mutant BCL9 HD2 and GST-ARD, as indicated; input lanes, 5% of the total binding reaction. C and D, co-immunoprecipitations of FLAG-tagged wt and mutant BCL9 or BCL9-2 with HA-β-catenin expressed in HEK 293T cells; bottom panels, protein expression levels in the lysates.