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. 2015 Mar 15;26(6):1044–1057. doi: 10.1091/mbc.E14-10-1438

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© 2015 Wei, Lin, 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: — The protein interaction between β-arrestin1/2 and GCN2. (A) A549 cells were cotransfected with YFP-tagged GCN2 and FLAG-tagged β-arrestin1/2. Immunofluorescence analysis was performed as described in Materials and Methods. Scale bar, 20 μm. (B) Cells were cotransfected with expression vectors harboring HA-GCN2 plus FLAG-β-arrestin1 (B-1) or FLAG-β-arrestin2 for 36 h (B-2) and untreated or treated with MG132 (20 μM) or ouabain (200 nM) for 8 h before being harvested. The cell lysates were immunoprecipitated with anti-HA IgG, and the immune pellets were detected by immunoblot analysis with anti-FLAG IgGs. (C) After treatment with 20 μM MG132, A549 cell lysates were used for immunoprecipitation with an anti-GCN2 IgG or normal rabbit IgG and analyzed by immunoblot analysis with anti–β-arrestin1/2 IgGs. (D) YFP-GCN2–transfected HEK293 cells lysates were precipitated with glutathione beads and incubated with purified GST, GST-β-arrestin1, or GST-β-arrestin2. Precipitates were analyzed by immunoblot analysis with an anti-YFP IgG. (E) Deletion mutants for GCN2 (E-1) and β-arrestin1/2 (E-2). (F) HEK293 cells were cotransfected with GCN2 deletion mutants plus FLAG-β-arrestin1 (F-1) or FLAG-β-arrestin2 (F-2). The cells were treated with MG132 (20 μM) for 5 h before being harvested, and lysates were immunoprecipitated with an anti-HA IgG, and the immune pellets were detected by immunoblot analysis using anti-FLAG IgG. (G) A549 cells were cotransfected with HA-GCN2 plus β-arrestin1 deletion mutants (G-1) or β-arrestin2 deletion mutants (G-2). The cells were treated with MG132 (20 μM) for 5 h before being harvested, and lysates were immunoprecipitated with anti-FLAG IgG; the immune pellets were detected by immunoblot analysis with anti-HA IgG. (H) HEK293 cells were cotransfected with HA-RWD, HA-PK1, HA-PK2, or HA-GCN2-N plus FLAG tagged β-arrestin1/2. The lysates were subjected to immunoblot analysis by using the indicated antibodies. A representative Western blot for each treatment from three independent experiments is shown.