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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: J Thromb Haemost. 2008 Jul 19;6(10):1789–1795. doi: 10.1111/j.1538-7836.2008.03088.x

Figure 4. Formation of a disulfide-linked GPIbβ dimer in the GPIb-IX complex containing the C484S/C485S mutation.

Figure 4

(A) GPIbβ in the mutant GPIb-IX complex is linked to another protein through a disulfide bond. Cell lysate from CHOαSSβIX cells was first co-immunoprecipitated with antibodies against GPIbα (Ibα lane), GPIX (IX lane) or mouse IgG (IgG lane) in the lysis buffer containing 1% digitonin, and then resolved in a SDS gel under non-reducing conditions. For comparison, 20% of the cell lysate used in immunoprecipitation was loaded directly in the same SDS gel ( lane). After transfer, the membrane was probed with a polyclonal anti-GPIbβ antibody. Note that only a band with a molecular mass of approximate 45kDa was present in the “Ibα” lane, and the apparent mass of GPIbβ monomer is approximately 25kDa. (B–D) GPIbβ in the mutant GPIb-IX complex is linked to another GPIbβ via a disulfide bond. CHOαSSβIX cell membrane including the embedded proteins was separated from the cytosolic fraction and co-immunoprecipitated by anti-GPIX antibody SZ1. The precipitated fraction was first resolved in a 4–12% Bis-Tris precast SDS gel (Invitrogen) under non-reducing conditions. The gel strip was either (B) directly stained by silver staining or extracted, incubated at 65 °C for 20 min in the SDS sample buffer containing 20% DTT and placed on top of a 12% Tris-glycine SDS gel for electrophoresis under reducing conditions. The proteins in the gel were either (C) visualized by silver staining or (D) transferred to a PVDF membrane and probed with anti-GPIbβ polyclonal antibody. The diagonal molecular weight markers in the 2-D gel were apparent in (C) and marked in (D). The dashed line highlights the positions of the 45-kDa protein complex in the 2-D gel. Silver staining showed that GPIbβ was the only protein in this 45-kDa complex. The protein complex with an apparent mass of approximate 150kDa in (B) is the antibody used for immunoprecipitation and used as the positive control for the 2-D gel. Each figure is a representative of at least 2 independent experiments.