Tyrosine phosphorylation of β-catenin mediated by Bcr-abl kinase diminishes β-catenin ubiquitination-proteosome–dependent degradation. (A) Western blot analysis of cell lysates from 293FT cells cotransduced with lentiviral vector (Lenti-Vect), lentiviruses carrying WT VE-cadherin (Lenti-VE-cad), retroviral vector (Retro-MiG), or retroviruses encoding p210-eGFP (Retro-MiG-p210). β-Catenin was densitometrically normalized to GAPDH with the control value set at 1.0, and all other values are shown relative to the control. (B) Immunoprecipitation of β-catenin was done using the above-mentioned cell lysates, and Western blots were probed with anti–P-Tyr (Phospho-Tyr102), anti–β-catenin, anti–phospho-Src, and anti-Src antibodies. IgG heavy chain served as the loading control. (C) DLR assays from the above 293FT cells transiently cotransfected with the phRL-TOP1 and phRL-TOP2 reporters and an internal pGL4.13-CMV-Luc2 firefly luciferase reporter. Data are shown as mean plus or minus SD (n = 3). The * denotes significant differences compared with vector controls (P < .01) based on the Student t test. (D) Western blot analysis of β-catenin stabilization from 293FT cells coexpressing VE-cadherin/eGFP-p210 or VE-cadherin/eGFP treated with 10 μM Src kinase inhibitor PP2, 10 μM Bcr-abl inhibitor imatinib mesylate, or both inhibitors for 16 hours. β-Catenin was densitometrically normalized to VE-cadherin with the untreated control value of expressing MiG vector alone set to 1.0. All other ratios are normalized to this control value. (E) VE-cadherin was immunoprecipitated from 293FT cells coexpressing VE-cadherin/eGFP-p210 or VE-cadherin/eGFP. The immunoprecipitates and cell lysates were loaded in parallel and probed with anti–phospho-Tyrosine (4G10) and VE-cadherin, c-Abl and eGFP antibodies. (F) β-Catenin immunoprecipitated either from 293FT expressing eGFP-210 or eGFP alone, or from Ph⁺ Sup-B15 or Ph⁻ REH leukemic cells was in vitro labeled with recombinant ubiquitin and subjected to in vitro degradation assay as described in “In vitro poly-ubiquitination of β-catenin.”