FIGURE 7.

Suppression of the constitutively active Vav2-Y172F mutant-mediated morphological transformation, activation of Rac1, and reorganization of junctional actin cytoskeleton by WT Cbl but not by E3-deficient mutants. A, whole cell lysates of MCF10A cells with stable expression of vector or Vav2-Y172F mutant were Western-blotted for Vav2. B, lysates of MCF10A cells with stable transduction of vector, wild type Cbl, or Cbl mutants were Western-blotted for Cbl. C, vector- or Vav2-Y172F-expressing MCF10A cells were stably transduced with vector, wild type Cbl, or Cbl mutants and selected with antibiotics for 2 weeks. The selected cells were trypsinized and replated at low density and cultured for 1 week. Cells were photographed with a phase-contrast light microscope under transmitted light. D–H, whole cell lysates of 16A5-Tet-On-172F cells with stable expression of vector, wild type Cbl, Cbl-Y700F, and Cbl-C3AHN (D) or stable expression of control shRNA, Cbl shRNA#2, and Cbl shRNA#4 (E) were immunoblotted for Cbl and β-actin. The cells were treated with vehicle or DOX for 3 days to induce Vav2 expression, and whole cell lysates were subjected to GST-PBD pulldown assay and immunoblotted for Rac1 (F and G). H, 16A5-Tet-On-172F cells with stable expression of vector, wild type Cbl, Cbl-Y700F, and Cbl-C3AHN were grown in DFCI media without EGF and treated with vehicle or DOX for 3 days. The cells were immunostained with anti-E-cadherin Abs (blue) and phalloidin (red). Both whole cell colony image and the high resolution image of the indicated areas in the colonies were acquired by confocal microscopy scanning at the subapical plane.