Analysis of GPIb and GPIX in 293T cells transfected with GPIb, GPIbβ, and GPIX. 293T cells were transiently transfected with the wild-type GPIb, GPIbβ, and GPIX or with the wild-type GPIb GPIX and mutant GPIbβ. The cells were then analyzed with an anti-GPIb polyclonal antibody, the anti-GPIX antibody FMC25 or the complex specific antibody AK1 (each figure is representative of four different experiments). (A) In the cells transfected with the wild-type GPIb, GPIbβ, and GPIX, there is a significant increase in fluorescence when the cells are reacted with the anti-GPIb polyclonal antibody (bold lines) compared with mock transfected cells (shaded area). When cells are transfected with the mutant GPIbβ and wild-type GPIb and GPIX, GPIb is detectable on the cell surface (thin line), but significantly reduced compared with the triple wild-type transfections. (B) When cells are transfected with the wild-type GPIb, GPIbβ, and GPIX, GPIX is readily detectable on the cell surface (bold line). However, there is no significant difference between the mock transfected cells (shaded area) and in cells transfected with the wild-type GPIb, GPIX, and mutant GPIbβ (thin line). Thus, GPIX is not detectable when the mutant GPIbβ is transfected with the wild-type GPIb and GPIX (thin line). (C) GPIb that is expressed on the cell surface of the wild-type triple transfection is recognized by the complex specific antibody AK1 (bold lines). In contrast there is no difference between the mock transfected cells (shaded area) and in cells transfected with the wild-type GPIb, GPIX, and mutant GPIbβ (thin line). Thus, GPIb that is expressed on the cell surface (A) in the transfections involving the mutant GPIbβ is not recognized by AK1, confirming the lack of surface expression of GPIX.