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. 2013 Jun 1;24(11):1776–1790. doi: 10.1091/mbc.E13-01-0026

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© 2013 Giridharan et al. 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: — Phosphatidic acid controls generation and trafficking through tubular recycling endosomes. (A) Metabolic pathways leading to phosphatidic acid synthesis. (B) PA levels were quantified enzymatically in HeLa cells treated with the indicated drugs for 1 h (compared with DMSO control treatment) from three independent experiments. Error bars, SE. (C) Cells treated as in B were immunoblotted for MICAL-L1, Synd2, and actin (loading control). (D) Cells treated as in B were immunostained for endogenous MICAL-L1 and Synd2. Bar, 10 μm. (E) Quantification of the percentage of cells from D with MICAL-L1 localized to tubular membranes from three independent experiments. Error bars, SE. (F–I) Cells incubated with Alexa Fluor 568–labeled transferrin for 20 min were either fixed (pulse) or fixed after 1 h treatment with the indicated drugs (chase). Bar, 10 μm. (J) HeLa cells incubated with Alexa Fluor 633–labeled transferrin for 30 min were fixed after 1 h treatment with DMSO (control) or the indicated drugs. The percentage of nonrecycled transferrin remaining in the cell was analyzed by flow cytometry from three independent experiments. Error bars, SE.