FIG. 2.
The 45-kDa K15 protein (aa 1 to 489) is associated with lipid rafts. Cos7 cells were transiently transfected with K15 expression construct K15 ex1-8 (A); the natural splice variant K15 ex1/6-8 (B), K15 ex1 as/4-8 (C), or K15 ex1 as/6-8 (D); or the LMP1-K15355-489 chimera (E). At 48 h after transfection, Cos7 cells were lysed in TNE buffer and extracts were analyzed on a flotation sucrose gradient as described in Materials and Methods. After ultracentrifugation, 1-ml fractions were collected starting at the top of the gradient and analyzed by Western blotting with a rabbit antibody to K15. Lipid raft-associated proteins are localized at the interface of 5 and 35% sucrose (fractions 4 and 5). Soluble proteins and solubilized membrane proteins of the nonraft plasma membrane are localized to the higher-density sucrose fractions (fractions 10 to 12, 35 to 42.5% sucrose). P, pellet. (A, top) The 45-kDa form of K15 ex1-8 (aa 1 to 489) localizes to lipid rafts (fractions 4 and 5, interface of 5 and 35% sucrose) and to the fractions containing solubilized membrane proteins (fractions 10 to 12, 35 to 42.5% sucrose). (A, bottom) Western blotting of the samples from the upper part of this panel probed with anti-caveolin 1. Endogenous caveolin 1 served as a positive control for lipid raft localization. (B) The 33- to 35-kDa protein derived from splice variant K15 ex1/6-8 is localized in the lipid raft fraction and high-density sucrose fractions (fractions 8 to 11). *, possible dimeric form. (C) The ∼21-kDa protein derived from splice variant K15 ex1 as/4-8 (see text) is found in lipid rafts and high-density sucrose fractions. (D) The ∼21-kDa protein derived from K15 ex1 as/6-8 is localized in lipid rafts and also in fractions containing solubilized membrane proteins (see text). (E) The LMP1-K15355-489 chimera is found in lipid rafts and high-density sucrose fractions.