Cellular localization and expression of 11kDa and NS1 in transfection. (A) Specificity of αNS1 and α11kDa polyclonal antibodies. UT7/Epo-S1 cells transfected with pGFP-NS1 or pGFP-11kDa were stained with respective antisera followed by a Texas red–conjugated secondary antibody. Images were taken from an Eclipse SE TE2000-S UV microscope (Nikon) at 20× magnification. (B) Cellular localization of GFP-NS1 and GFP-11kDa in transfected UT7/Epo-S1 cells and CD36⁺ EPCs. UT7/Epo-S1 cells and CD36⁺ EPCs were transfected with pGFP-NS1 or pGFP-11kDa and stained with DAPI at 48 hours after transfection. DAPI was used to stain the nuclei. The confocal images in panels B and C were taken at 60× (objective lens) magnification with an Eclipse C1 Plus confocal microscope (Nikon). (C) Cellular localization of 11kDa and NS1 in B19V-infected CD36⁺ EPCs. Infected CD36⁺ EPCs (at 48 hours after infection) were stained with α11kDa and αNS1 antisera followed by a Texas red–conjugated secondary antibody, respectively. DAPI was used to stain the nuclei. (D) The protein levels of GFP-NS1 and GFP-11kDa in transfected UT7/Epo-S1 cells and CD36⁺ EPCs versus the NS1 and the 11kDa expressed in B19V-infected CD36⁺ EPCs, respectively. UT7/Epo-S1 cells and CD36⁺ EPCs were transfected with either pGFP-11kDa or pGFP-NS1 and stained at 48 hours after transfection. CD36⁺ EPCs were infected with B19V and stained at 48 hours after infection. Cells were fixed with 1% paraformaldehyde and permeabilized in 0.2% Tween-20. Either α11kDa or αNS1 antiserum at a dilution of 1:100 was used to immunostain cells, followed by a Cy5-conjugated secondary antibody. Stained cells were analyzed by flow cytometer. The protein level, represented by the mean fluorescence intensity, was compared between transfected and infected cells. S1 indicates UT7/Epo-S1; CD36⁺, CD36⁺ EPCs; and TX, transfection.