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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: J Thromb Haemost. 2009 Jun 29;7(9):1533–1540. doi: 10.1111/j.1538-7836.2009.03536.x

Figure 2. Both ectodomain and transmembrane domain of GPIbβ are required for expression of GPIX in CHO cells.

Figure 2

(A) Illustration of GPIbβ and GPIX constructs used in the study. An HA epitope tag was attached to the N-terminus of either full-length GPIbβ or the isolated GPIbβ ectodomain (IbβE) for detection. HA-IbβE and IXE contain only the ectodomain of GPIbβ and GPIX, respectively. (B) Production of GPIbβ and GPIX proteins in transfected CHO cells. After transient transfection, cells were lysed by 1% Triton X-100 lysis buffer, and proteins were separated in a 12% Tris-glycine SDS gel, transferred to PVDF membrane and immunoblotted by HRP-conjugated anti-HA antibody (for HA-Ibβ), goat anti-GPIX polyclonal antibody (for GPIX and IXE), or anti-actin antibody. Each lane is identified on top by the subunits transfected into the CHO cell. Cells transfected with empty vector pDX were included as a negative control for GPIX detection (** lane). Each gel in this figure is a representative of 3-5 independent experiments. (C) Overlaid histograms showing surface expression levels of GPIX, in the presence of noted GPIbβ variants, as measured by flow cytometry after immunostaining with anti-GPIX antibody FMC25. The expression level of GPIX in CHO cells transfected with GPIb-IX complex (GPIbα/HA-Ibβ/GPIX) and empty vectors (pDX) were shown as positive and negative controls, respectively. The plot is a representative of no less than 4 independent experiments. (D) Secretion of HA-IbβE and lack of secretion of IXE in the culture medium. Proteins in the culture medium of various transiently transfected cells were precipitated by anti-HA antibody (for HA-IbβE) or FMC25 (IXE), separated in a 12% Tris-glycine SDS gel, transferred to PVDF membrane and blotted by HRP-conjugated anti-HA antibody or goat anti-GPIX polyclonal antibody. As a positive control for detection of GPIX (* lane), CHO cells transiently transfected with GPIbα/HA-Ibβ/GPIX were lysed, and GPIX in the lysate was immunoprecipitated and blotted as the other samples.