Identification of the β-catenin binding site in the NH₂-terminus of BCR and p210 BCR/ABL. (A) Whole-cell lysates (WCLs) were collected from K562 cells and used for affinity precipitations (APs) using agarose-conjugated immunoglobulin G (IgG-Ag) or agarose-conjugated ubiquitin (Ub-Ag). Precipitated proteins were resolved and identified by western blot by using an antibody that recognizes the NH₂-terminus of BCR and p210 BCR/ABL (BCR/ABL). (B) Yeast 2-hybrid analysis was used to map the UBD to residues 180-191 of BCR and p210 BCR/ABL. The upper schematics show the domain structure of the full-length BCR and p210 BCR/ABL constructs that were used for the study (shaded area indicates the ABL sequences). The solid lines below indicate the predicted translational products of the truncation mutants that were used to map the UBD. Binding is indicated as plus (+) or minus (–). (C) 293T cells were transfected with p210 BCR/ABL (BCR/ABL), the ΔUBD mutant, or cognate vector (vector). Lysates were collected and examined by western blot for expression (WCL, lower panel) or used for affinity precipitations using agarose-conjugated ubiquitin (upper panel). Affinity-precipitated BCR/ABL was normalized to input and then expressed as a percentage of wild-type BCR/ABL (Ub affinity). (D) WCLs were collected and examined by western blot for expression of the constructs (BCR/ABL), autophosphorylated p210 BCR/ABL (p-BCR/ABL), CrkL, or phosphorylated CrkL (p-CrkL) as indicated. (E) WCLs were examined by western blot for expression of the transfected constructs or endogenous GRB2. Lysates were used for immunoprecipitations (IPs) using a GRB2 antibody, and immunoprecipitates were examined by western blot using antibodies for p210 BCR/ABL (BCR/ABL) or GRB2 as indicated. (F) WCLs were examined by western blot for expression of the transfected constructs, total ERK1/2, and activated ERK1/2 (p-ERK1/2) as indicated. All data shown represent the average of at least 3 independent experiments. P values were calculated by using a paired Student t test or a one-way analysis of variance (ANOVA) followed by Tukey tests. O, oligomerization domain; GEF, guanine nucleotide exchange factor domain; C2, C2 domain; GAP, GTPase-activating domain. *P < .05; **P < .01; ***P < .001.