Characterization of L929 transfected cells.

(A) Expression of CD146 was analyzed by flow cytometry. The 2 independent clones of CD146-transfected cells in sense orientation, B9-5 and L4B11E9, expressed CD146 at different levels, whereas the CD146-transfected clone in antisense orientation, D12, did not express CD146. (B) In B9-5 cell lysate (lane b), Western blotting revealed a band with a molecular weight identical to in HUVEC lysate (lane a) at 120 kd, whereas no band was observed in D12 cell lysate (lane c). (C) Determination of the paracellular permeability coefficient showed a significant decrease for B9-5 and L4B11E9 cells compared with D12 cells (P = .02; n = 6). When EGTA (5 mM for 30 minutes at 37°C) was added, a retraction of transfected cells was observed, thereby excluding the possibility of measuring the permeability coefficient after a reduction in calcium concentration.