Stromal cells up-regulate VE-cadherin expression and stabilize β-catenin in Ph⁺/VE-cadherin⁺ leukemic cells. (A) Western blot analysis of Sup-B15 cells in LTMC (Med) or in LTCC with either S10 (S10) or PatX (PatX) stromal cells. The same membrane was probed with anti–VE-cadherin, anti–β-catenin, and anti-Src, respectively. GAPDH was used as the lane loading control. (B) Western blot of cell lysates isolated from Sup-B15 cells exposed to recombinant human cytokines, chemokines, or growth factors compared with untreated control cells (Cont). (C) Reciprocal coimmunoprecipitation of Sup-B15 cell lysates from LTMC (Med) or LTCC (S10 and PatX) with specific antibodies for anti–VE-cadherin or anti–β-catenin. Membranes were reciprocally probed with anti–β-catenin and anti–VE-cadherin, respectively. IgG heavy chains served as the loading control. Input denotes sample with antibody alone, lacking cell lysate. (D) Western blot analysis of cell lysates from 293FT (top) or Sup-B15 cells (bottom) transduced with lentiviruses carrying the wild-type VE-cadherin (WT) or VE-cadherin lacking either the cytoplasmic domain (Δcyto) or β-catenin binding domain (Δbcat) CDSs (coding DNA sequences). Vect denotes the empty lentiviral vector encoding the blasticidin resistance protein without a CDS insert. (E) Western blot analysis of Sup-B15 cells transiently transfected with VE-cadherin siRNA or scrambled (Scr) control siRNA sequence. Time-course (top) and dose-response (bottom) of VE-cadherin siRNA-transfected samples were probed with specific antibodies for anti–VE-cadherin and β-catenin.