VWF-deficient cells display reduced β3 integrin expression and αvβ3-dependent adhesion, increased rate of β3 integrin internalization, and elevated release of Ang-2. (A) β3 mRNA expression was measured by RT-PCR. (B) Total β3 integrin protein expression was measured by Western blotting (representative blot shown) and quantified by densitometry relative to tubulin. (C) Surface levels of total αvβ3 (antibody: LM609) were measured by flow cytometry at 24 and 48 hours after siRNA treatment. (D) Control or siVWF-treated cells, 48 hours after transfection, were seeded onto different extracellular matrix substrates. After 40 minutes, nonadherent cells were removed by gentle washing and the number of bound cells was quantified relative to the total number seeded. (E) Internalization of β3 integrin from the cell surface after incubation at 37°C for 7.5 minutes. (F) Levels of Ang-2 in the supernatant of control or siVWF-treated cells were measured 48 hours after transfection by ELISA. Data (A-C, F) were normalized to control siRNA-treated cells at each time point. Open bars, siCTL; closed bars, siVWF. Error bars indicate mean ± SEM (n = 3). *P < .05; **P < .01 (Student t test).