Immunofluorescence staining for VIIa or TF.

BHK(TF) cells treated with 10 nM VIIa (A-C, G) or buffer (D-F) were immunofluorescence stained with VII antibodies (A-E) or TF antibodies (F, G). (A) Cells were treated with 10 nM VIIa for 1 hour at 37°C, and the unbound VIIa was removed before fixing the cells. (B) Cells were treated with 10 nM VIIa for 1 hour at 37°C, the unbound VIIa was removed, and the cell surface–associated VIIa was eluted with low-pH buffer to illustrate the cell surface–internalized VIIa. (C) Cells were treated with 10 nM VIIa for 1 hour at 4°C, the unbound VIIa was removed, and the cell surface–associated VIIa was eluted with low-pH buffer. (D, E) Cells were treated the same as in panels A and B, respectively, except that VIIa was not included in the incubation medium. (F, G) Immunostaining of TF after 1-hour exposure of BHK(TF) cells at 37°C to the control medium and 10 nM VIIa, respectively.