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. 2010 Dec 1;21(23):4141–4150. doi: 10.1091/mbc.E10-05-0424

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 5. — OSBP depletion reduces the cholesterol content of Golgi/endosomal membranes containing PI4KIIα. (A) The localization of cholesterol was determined by filipin staining of CHO cells transiently expressing PI4KIIα-GFP and treated with or without 25-hydroxycholesterol (2.5 μg/ml) for 60 min. (B) Equivalent amounts (2–2.5 mg protein) of the postnuclear supernatants from CHO cells expressing shOSBP or a nontargeting control (shNT) were fractionated on an Opti-Prep gradient and assayed for unesterified cholesterol content as described in Materials and Methods. The cholesterol content of individual fraction is expressed as a percentage of the total cholesterol recovered from the gradient and is the mean and SEM of three experiments (*p < 0.05). (C) Distribution of the organelle markers NPC1 (late endosomes), TGN38 (Golgi), VAP (ER), and caveolin (plasma membrane/endosomes), as well as OSBP and PI4KIIα, were determined by immunoblotting of equivalent volumes of each fraction.