VWF/ristocetin activates PI3-kinase in GPIbα-expressing cells. (A) Flow cytometry for the surface expression of GPIbα on CHO cells expressing either wild-type GPIbα or GPIbα deletion mutants using the anti-GPIbα monoclonal antibody, WM23, and a FITC-labeled secondary antibody.²⁶  The solid histogram is the secondary antibody alone. (B) CHO cells expressing either wild-type GPIbα or mutant GPIbα were incubated with VWF/ristocetin for the indicated times. Cells were then lysed, blotted with either anti-Akt or anti–phospho-Ser308 Akt antibodies, and visualized as described in “Methods.” The results are representative of 3 separate experiments. Vertical lines indicate repositioned lanes from the same experiment and Western blot. (C) Binding of FITC-labeled VWF (10 μg/mL, final concentration) in the absence (solid histogram) or presence (open histogram) of ristocetin (1 mg/mL, final concentration) to CHO cells expressing wild-type GPIbα or GPIbα containing deletion mutations. CHOβIX cells (βIX) lacking GPIbα were used as a negative control.