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. 2012 May 15;23(10):1860–1873. doi: 10.1091/mbc.E11-09-0746

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© 2012 Orsi 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: — mAtg9 is on recycling endosomes. (A) Homogenates from HEK293 cells were separated on 1–22% Ficoll gradients. Gradient fractions were loaded from left (heavy) to right (light) and then analyzed by Western blot using antibodies against CI-MPR (marker for late endosomes/TGN), EEA1 (early endosomes), TGN46 (TGN), mAtg9, TfR (RE), and SOD1 (cytosol). (B) Distribution of marker proteins. The intensity of each band (in arbitrary units) is plotted on the y-axis and the fraction number on the x axis. mAtg9, black solid line; all other markers are dashed lines: red, CI-MPR; yellow, EEA1; green, TGN46; blue, TfR; gray, SOD1. (C) Lysates from HEK293 cells incubated in full medium (F) or starved for 2 h (S) were immunoprecipitated using beads alone (–), a nonrelevant antibody (NR) at 1× and 2× immunoglobulin G (IgG), and anti-mAtg9 IgG and then analyzed by Western blot using antibodies against mAtg9 and TfR. Input, 5% of total lysate before immunoprecipitates (IP). Data are representative of three independent experiments.