Cell-surface expression and localization of CD47 in HUVECs. (A) HUVECs were treated with medium or medium containing TNF-α (left), IL-1β (middle), or IL-1β and IFN-γ (right) for 4 hours and 24 hours, and CD47 expression was detected by flow cytometry using anti-CD47 (B6H12) mAb and PE-labeled anti–mouse secondary mAb. An isotype-matched mAb was used as a negative control. (B) Untreated (top) and TNF-α treated (bottom) nonpermeabilized HUVECs were fixed with 2% paraformaldehyde and stained with anti-CD47 (B6H12; left; 10 μg/mL) or anti–VE-cadherin (Hec 1; right) mAb as described in “Immunofluorescence microscopy.” Bars, 40 μm.