Fig 13.
Inhibition of ADAM17 decreased the production of the 75-kDa VEGFR2 fragment. (A) Cultured mouse Cdc42flox/flox ECs were infected with Ad-Cre or Ad-LacZ and were then incubated with or without TAPI-1 (50 μM) or INCB3619 (2 μM) for 4 h. Cell lysates were reacted with antibodies for cleaved caspase 3 (top), Cdc42 (center), or vinculin (bottom). (B) The intensities of bands from three independent experiments were quantitatively analyzed. *, P < 0.05. (C) HUVECs were either left untreated or treated with PMA (25 ng/ml) for 1 h and were then analyzed by Western blotting using antibodies against VEGFR2 and vinculin. (D) The intensities of bands from three independent experiments were quantitatively analyzed. (E) HUVECs transfected with Cdc42 siRNA, alone or together with ADAM17 siRNA, were incubated with or without VEGF (50 ng/ml) for 30 min and were then subjected to an internalization assay (top). The cell lysates from each sample were analyzed by Western blotting with antibodies against Cdc42, ADAM17, and vinculin, as indicated. (F) The relative intensities of 75-kDa VEGFR2 fragments were quantitatively analyzed. (G) HUVECs transfected with scrambled siRNA, Cdc42 siRNA alone, or Cdc42 siRNA together with ADAM17 siRNA. Cellular membrane and cytosol fractions were separated and were then blotted with various antibodies as indicated.