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. Author manuscript; available in PMC: 2017 Jul 17.
Published in final edited form as: J Neurosci Res. 2009 Nov 15;87(15):3369–3377. doi: 10.1002/jnr.22099

Fig. 4.

Fig. 4

Isolation of detergent (Lubrol WX)-resistant membranes from OPCs. A: OPCs were lysed and solubilized in 0.5% Lubrol WX at 4°C. Lysates were mixed with 80% sucrose and subsequently overlaid with 35% and 5% sucrose. Sucrose gradients were centrifuged at 46,900 rpm for 18 hr at 4°C to separate buoyant raft membranes from bulk cellular material. Fractions 1–9 (equal volume) were taken from the top of each gradient and were used for SDS-PAGE and Western blot analysis. Flotillin-1, a marker for raft microdomains, was present in both buoyant “rafting” (2–3) fractions. GM-1 ganglioside, a raft marker, was detected in fractions 1 and 2 (GM-1 standard shown). Calnexin was localized only within nonbuoyant fractions. IGF-IR, p85, and Akt were detected strongly in nonbuoyant fractions but were also seen in buoyant fractions. B: Total cholesterol and protein distributions across fractions analyzed in A were determined as described in Materials and Methods. Squares (dashed line) represent total protein; lozenges (solid line) represent total cholesterol in each fraction. The majority of cholesterol is contained within the buoyant fractions. In contrast, the amount of protein per fractions is much greater in the nonbuoyant fractions. C: OPCs serum starved for 2 hr and treated or not with IGF-I (20 ng/ml) for 15 min were lysed and solubilized in 0.5% Lubrol WX at 4°C. Lysates were centrifuged at 13,000g for 20 min to separate detergent insoluble (caveolin-1- and cholesterol-enriched) fractions from the soluble supernatant. Equal protein fractions were analyzed by SDS-PAGE and Western blot analysis. Phosphorylated Akt and IGF-IR were increased in the insoluble fractions with IGF-I stimulation. Total Akt, IGF-1R, and p85 are also detected in insoluble fractions and were unchanged with IGF-I treatment. D: Total cholesterol (lozenges and solid line) and protein (squares and dashed line) distribution in detergent insoluble and soluble fractions that correlate with fractions analyzed in C. Most cholesterol is contained within the Lubrol WX-insoluble fractions.