BSS mutations in GPIbβ_E disrupt expression and assembly of GPIb-IX complex by different mechanisms. (A) Two views of the GPIbβ_E structure are shown as a ribbon diagram related by a 90-degree rotation. Residues affected by BSS missense mutations are highlighted as stick and colored green for being solvent accessible and red for buried. (B) Space-filling representation of the GPIbβ_E structure, showing the concave (left) and convex (right) faces. Main chain atoms are colored white, and residues affected by BSS mutations are colored green. (C) SDS gels showing differential effects of BSS mutations on expression (top), secretion (middle), and folding (bottom) of GPIbβ_E expressed from transfected CHO cells. Each mutation is identified by the residue number. The HA epitope tag was appended to the N-terminal end of GPIbβ_E for easy detection. Immunoprecipitation was performed with anti-HA monoclonal Ab and immunoblotting with HRP-conjugated anti-HA monoclonal Ab (HRP-HA). Molecular weight markers are marked on the left of each gel. (D) Sample flow cytometric histograms showing the effects of selected BSS mutations on surface expression levels of HA-tagged full-length GPIbβ (HA-GPIbβ) and GPIX (GPIX) that are coexpressed transiently in CHO cells. (E) Relative surface expression levels of HA-GPIbβ (gray column) and GPIX (white column) in transfected CHO cells. The surface expression levels were measured by flow cytometry and quantified as mean fluorescence intensity, which were normalized with expression levels in cells transfected with wild-type GPIb-IX (GPIbα/HA-GPIbβ/GPIX) being 100% and those in cells transfected with sham vectors 0%.¹⁷  The data are presented as mean ± SD (n = 3). *P < .001.