FIGURE 4.

Isolation of raft and non-raft microdomains from SH-SY5Y-wt cells. A, SH-SY5Y-wt cells were incubated with 100 μm DHA, solubilized in Triton X-100, and subjected to sucrose gradient fractionation for the isolation of raft and non-raft microdomains. The cholesterol levels of the collected fractions of the sucrose gradient were analyzed by a H₂O₂ Amplex Red-based assay. DHA-incubated cells show a significant increase in the ratio of non-raft/raft cholesterol, indicating that DHA displaces cholesterol out of the raft microdomains. B, same fractions were subjected to the γ-secretase activity assay. A similar shift in the ratio of non-raft/raft γ-secretase activity and PS1 protein levels were observed in the presence of DHA compared with cells treated with solvent control. Gradient fractions were also analyzed for β-secretase activity and BACE1 protein levels. C, representative WB analysis of the collected gradient fractions for the proteins flotillin, cadherin, PS1, and BACE1. Each fraction was analyzed by SDS-PAGE and immunoblotted with the respective antibodies. Flotillin-positive fractions represent detergent-insoluble raft fractions. Cadherin-positive fractions represent detergent-soluble non-raft microdomains. In the presence of DHA, PS1 protein level was reduced in flotillin-positive fractions.