FIG 8.

Effect of pretreating U251 cells with SCARB2 antibodies on the binding of EV71 to the cells. (A) Flow cytometry analysis of the cell surface expression of U251, RD, Vero, HeLa, and Jurkat cells. Cells were fixed and incubated with either mouse IgG (black line) or antibody to vimentin (gray line) and then incubated with the fluorescent secondary antibody and subjected to flow cytometry analysis. y axis (counts) = cell counts; x axis = fluorescence density. (B) Quantitative RT-PCR analysis of the binding of EV71 to VK-U251 cells that were infected after preincubation with antibodies to either vimentin (Vim ab; 60 μg ml⁻¹), PSGL-1 (PSGL-1 ab; 60 μg ml⁻¹), SCARB2 (SCARB2 ab; 60 μg ml⁻¹), or isotype IgG (isotype ab; 60 μg ml⁻¹). SCARB2 ab + Vim ab = cells preincubated with SCARB2 antibody (60 μg ml⁻¹) for 30 min at 37°C and then incubated with vimentin antibody for 30 min at 37°C. Vim ab + SCARB2 ab = cells preincubated with vimentin antibody for 30 min at 37°C followed by incubation with SCARB2 antibody for 30 min at 37°C. Control, EV71-infected cells without preincubation; mock, mock-treated cells with no virus. (C) Quantitative RT-PCR analysis of the binding of EV71 to U251 cells that were preincubated with antibodies to either vimentin, SCARB2, or isotype IgG as described above. (D) Determination of EV71 virus titers in infected U251 cells and the corresponding culture media at 12, 24, and 48 h after infection. The cells were preincubated with vimentin antibody (Vim ab; 60 μg ml⁻¹), SCARB2 antibody (SCARB2 ab; 60 μg ml⁻¹), or isotype IgG (control; 60 μg ml⁻¹) before infection. Mock, uninfected cells. (E) Determination of EV71 virus titers in infected VK-U251 cells and the corresponding culture supernatants as described for panel D. Cells were preincubated with vimentin antibody (Vim ab), SCARB2 antibody (SCARB2 ab), or isotype IgG (isotype ab) before infection. Control, EV71-infected cells without preincubation.