FIG. 8.

A portion of EhLimA associates with lipid rafts. (A) Total cell lysates prepared with the addition of cold Triton X-100 were subjected to centrifugation resulting in soluble (S) and insoluble (P) fractions. A Western blot analysis using anti-EhLimA antibodies shows that a significant portion of EhLimA is found in the Triton X-100-insoluble fraction. A similar protein distribution of the amoebic Gal-lectin light subunit (Ehlgl) was detected using polyclonal anti-Ehlgl antibodies. (B) Total cell lysates prepared with cold Triton X-100 were subjected to sucrose gradient flotation. Twelve fractions were collected from the top of the gradient and proteins were precipitated with trichloroacetic acid. Following SDS-PAGE and immunoblotting, the distribution of EhLimA, Ehlgl, and actin in the fractions was analyzed. A portion of EhLimA as well as of Ehlgl and actin was detected in the lower-density regions of the gradient. The presence of EhLimA in the lower-density regions was observed in three independent experiments. The percentage of sucrose in each fraction is indicated. (C) Depletion of cholesterol. Cells were incubated for 30 min with either digitonin or methyl-β-cyclodextrin on ice followed by lysis in Triton X-100 and centrifugation. Western blotting reveals that the distribution of EhLimA between soluble (S) and insoluble (P) fractions was greatly affected by digitonin but not by methyl-β-cyclodextrin.