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. 2011 Apr 20;286(24):21315–21323. doi: 10.1074/jbc.M110.202549

FIGURE 2.

FIGURE 2.

The GP Ib-IX complex associates with the GEMs of platelets and CHO cells. A, resting human platelets were lysed with cold 0.1% Triton X-100 or 1% Brij 35. Lysates were subjected to sucrose gradient density centrifugation. Equal volume aliquots of each fraction (total 12 fractions) were analyzed by reducing SDS-PAGE followed by Western blotting with anti-GP Ibα monoclonal antibody WM23, anti-GP Ibβ, and anti-GP IX polyclonal antibodies. Blots represent three independent experiments. The GEM fraction floats within sucrose gradient fractions 1–4, and the position of the GEMs was identified by blotting flotillin-1. B, CHO cells expressing the GP Ib-IX complex were lysed with 0.5% Brij 35 or 0.1% Triton X-100 and subjected to sucrose gradient density centrifugation. The localization of GP Ibs in the density gradient was revealed by the same antibodies as A. The CHO GEM markers used are flotillin and caveolin. The non-GEM marker used is CD71. C, the association of the GP Ib-IX complex with the GEMs on CHO cells is cholesterol-dependent because saponin (0.5%/g/v), a cholesterol-depriving chemical, depleted the majority of the GP Ib-IX complex and caveolin from the GEMs.