Colocalization of internalized FVIIa and Rab GTPases.

<p>CHO-EPCR cells were exposed to AF488-FVIIa (50 nM) for 1 h at 4°C. At the end of 1 h, the supernatant was removed, cells were washed quickly with Ca²⁺/Mg²⁺-containing buffer to remove the unbound ligand and then transferred to 37°C to induce internalization of the surface bound ligand. At the end of 0, 5, and 15 min, cells were fixed, permeabilized, and immunostained with mouse monoclonal or rabbit polyclonal antibodies against EPCR, Rab5A, Rab4A, or Rab11 followed by Rhodamine Red-labeled anti-mouse/anti-rabbit IgG as a secondary reporter antibody. The cells were imaged as described in methods. Left panel, immunostaining of EPCR, Rab5A, Rab4A or Rab11; middle panel, fluorescence of AF488-FVIIa. The right panel depicts the merged images of left and middle panels. The insets show the magnified view of the boxed regions.