gp96/grp94 chaperones the GPIb-IX complex. (A) Knockdown of gp96/grp94 results in concomitant loss of GPIbα surface expression. Flow cytometric staining was performed for surface GPIbα (AK2), followed by intracellular staining for gp96/grp94 in Ib9 cells. Data are representative of 5 independent experiments. (B) gp96/grp94 is important for the stable expression of multiple subunits of the GPIb-IX complex. Individual components of the complex were blotted from the total cell lysate of Ib9 cells transduced with either EV or gp96/grp94 shRNA lentivector. (C) Differential glycosylation of GPIbα in the absence of gp96/grp94 as shown by Endo-H treatment and Western blot for GPIbα in EV and gp96/grp94 shRNA Ib9-IX cells. (D) The GPIX subunit is degraded in the absence of gp96/grp94. Western blot analysis for GPIX subunit expression in EV and gp96/grp94 shRNA-treated Ib9-IX cells and in gp96/grp94 shRNA Ib9-IX cells after treatment with the inhibitors kifunensine (KIFU) and chloroquine (CLQ). Vertical lines have been inserted to indicate a repositioned gel lane. Data are representative of 2 independent experiments. (E) Coimmunoprecipitation of GPIX and gp96/grp94 in CHO-Ib9/IX cells (Ib9/IX). GPIX-FLAG or gp96/grp94 proteins were immunoprecipitated, followed by immunoblotting. Isotype control antibodies are included. (F) Recombinant GST-gpIX or GST was incubated with mouse gp96/grp94, followed by GST pull-down and immunoblot for gp96/grp94 and GST, respectively.