Lipid-raft association of stomatin after TX-100 lysis changes on mβCD-treatment.

Platelets (2 × 10⁹ cells/mL) were incubated for 30 minutes at 37°C in the presence (mβCD-treated) or absence (untreated) of 0.5% mβCD, washed twice in buffer I containing 2 mM CaCl₂, and lysed in 1% TX-100 at 4°C. The lysates containing 5.3 mg/mL protein were adjusted to 50% sucrose, and 750 μL was transferred to SW 50 centrifuge tubes; overlaid with 40%, 30%, 20%, and 10% sucrose (750 μL each); and centrifuged for 17 hours at 230 000g. Five fractions (750 μL) were collected from the top, and the pellet (P) was resuspended in 750 μL TBS. Aliquots were analyzed by silver staining (A) and Western blotting (B) and for cholesterol content (the concentration of cholesterol is given in nanomoles per milliliter). Equal volumes of the fractions were loaded on the gels except for fraction 5 in panel A, which was diluted 1:10 in sample buffer. Arrowheads in panel A indicate the respective protein bands of fraction 2, which were identified by MALDI-MS. Bars in panel A indicate molecular-weight markers of 205, 116, 97, 66, 45, and 29 kd. Representative data are shown (n = 4).