Fig. 1. Effect of domain deletion in GPIX on expression and assembly of the GPIb-IX complex in transiently transfected CHO cells.

(A) Illustration of mutant GPIX constructs. (B) Surface expression levels of GPIbα (gray bar) and GPIX (white bar) in mutant cells. The vector containing wild type or mutant GPIX cDNA was co-transfected transiently with wild type GPIbα and GPIbβ cDNAs into CHO cells. After 2 days, the cells were harvested, stained by antibodies against GPIbα (AK2) or GPIX (FMC25), and analyzed by flow cytometry. The cells were identified by the subunits transfected. “α” stands for GPIbα, “β” GPIbβ, and “IX” GPIX. The subscript denotes the mutation within the subunit. The mean fluorescence intensity (MFI) were normalized such that the level in wild-type CHOαβIX cells is 100% and that in sham vector transfected CHOpDX cells 0%. The data are calculated from 3 independent experiments and presented as the mean ± s.d.. (C) Overall expression of GPIbα and GPIX in transfected CHO cells. Cell lysates were resolved in reducing SDS gels and transferred to the PVDF membrane. The membrane was probed separately with antibodies against GPIbα (WM23), GPIbβ (Gi27), GPIX (polyclonal), and actin. Band intensities for each complex subunit were quantitated and expressed as a percentage of the wild type level. The numbers below the bands are the average of 3 independent experiments, and the deviation is usually 10%. (D) Effect of domain deletion in GPIX on formation of the GPIbα-GPIbβ disulfide bonds (i.e. GPIb formation). Cell lysates were resolved in non-reducing SDS gels and transferred to PVDF membrane. The membrane was probed with Gi27 first, stripped and re-probed with WM23. The blots are representatives of 2 independent experiments. The membrane was also probed directly with WM23 in 3 additional independent experiments (not shown). The GPIb and non-GPIb bands are indicated on the right.