TM4SF5-mediated VEGF expression involves retention of cell-surface integrin α₅. (A,B) SNU449 cells were transiently transfected with a mock construct or pcDNA3-TM4SF5 for 48 hours. (A) Transfected cells were collected, and an equal number of cells were probed with antihuman integrin α₅ (P1D6) and antimouse IgG-conjugated with phycoerythrin without (left) and with (right) permeabilization, before fixation and flow cytometric analysis. Note that the histogram for integrin α₅ on the surface of TM4SF5-transfected cells (TM4SF5) without permeabilization is right-shifted, compared with that of mock construct-transfected cells (Mock). Control indicates no incubation with the primary antibody. (B) Transfected cells were suspended and allocated into sets with an equal number of cells, which were treated with trypsin at the indicated units for 15 minutes at room temperature. Cells were then mixed with 1 mM phenylmethylsulfonyl fluoride in PBS, washed with PBS, and lysed at 4°C, before standard Western blots for the indicated molecules. (C,D) Conditioned media were collected from SNU449Tp cells that had been suspended and incubated with either normal mouse IgG or anti–human integrin α₅ (P1D6) monoclonal antibody (30 μg/mL) 20 minutes before reseeding onto normal culture dishes for 10 hours of incubation. (C,F) Whole-cell lysates were prepared and immunoblotted for the indicated molecules. (D) Conditioned media were analyzed for VEGF levels using the angiogenic antibody array, as in Figure 2D. Dotted rectangles represent array blots for VEGF. Data shown are representative of 3 different experiments. (E,F) TM4SF5-null SNU449 (E) or SNU398 (F) cells were transiently cotransfected with various plasmids. Immunoprecipitates using anti-(His)₆ antibody and lysates (WCL) were blotted in parallel. α₅ecto or α₅cyto indicates α₅ integrin that was immunoblotted by the antibody recognizing an extracellular region or the cytoplasmic domain of integrin α₅, respectively. X4C5 expression was much less than integrin α₅ or α_5/1, but X4C5 showed increased coimmunoprecipitation with myc-(His)₆-TM4SF5 than α_5/1. *Nonspecific band for an internal control. Data shown are representative of 3 isolated experiments.