Fig. 4.

ABCA1 resides in a distinct membrane domain. A: Normal HSFs (N-5) grown to subconfluent stage in 100 mm dishes (one dish per treatment) were treated with the indicated detergents, as described in the Materials and Methods. Cells were homogenized, and the PNS obtained was spun at 100,000 g for 1 h. The resultant DRM pellet (R) and detergent-soluble supernatant (S) were subjected to immunoblot analysis for ABCA1, caveolin-1 (Cav-1), calnexin, and LAMP-2. The detergents used are as follows: 1.0% Triton X-100 (TX100), 1.0% Triton X-102 (TX102), 1.0% Brij 58, 1.0% Brij 98, 1.0% Lubrol WX (Lubrol), 1.0% CHAPS, 2.0% CHAPSO, and 1.0% Tween 20. B, C: Normal HSFs (N-5) (B) or differentiated THP-1 cells (C) were treated with 1.0% TX100 or with 1.0% Brij 98 as above, and PNS was subjected to density gradient centrifugation by using OptiPrep, as described in the Materials and Methods. Eight 0.5 ml fractions collected from the top were subjected to immunoblotting with antibodies to ABCA1, Cav-1, flotillin-1 (Flot-1), or LAMP-2.