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. 2020 Dec 1;31(25):2816–2825. doi: 10.1091/mbc.E20-03-0173

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© 2020 Li et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 4: — Grp1 recruitment to the recycling endosome involves both lipid-based and protein-based mechanisms. Quantitative results are shown as mean with standard error: *, p < 0.05, NS p > 0.05, Student’s t test. The number of independent experiments performed is specified below. (A) Reconstituting the recruitment of Grp1 to liposomal membrane. Liposomes were endowed with different factors as indicated, and then incubated with full-length Grp1-T280D. The degree of recruitment was then quantified; n = 3 experiments. (B) Recruitment of different forms of Grp1 to liposomal membrane. Constructs as shown were incubated with fully endowed liposomes (containing PI4P, GRASP, and IPCEF1), followed by centrifugation to detect distribution in the pellet (P) vs. supernatant (S); FL (full-length Grp1-T280D), CC-Sec7 (Grp1 lacking the PH domain), Sec7-PH (Grp1-T280D lacking the CC domain). A representative result along with quantitation from three experiments is shown. (C) Recruitment of the CC-Sec7 construct to liposomal membrane requires GRASP/IPCEF1. The CC-Sec7 construct was incubated with liposomes that were endowed with different factors as indicated, followed by centrifugation to detect distribution in the pellet (P) vs. supernatant (S). A representative result along with quantitation from three experiments is shown. (D) Recruitment of the Sec7-PH construct to liposomal membrane requires PI4P. The Sec7-PH construct was incubated with liposomes that were endowed with different factors as indicated, followed by centrifugation to detect distribution in the pellet (P) vs. supernatant (S). A representative result along with quantitation from three experiments is shown. (E) Recruitment of different forms of Grp1 to the recycling endosome. Constructs as shown were stably transfected into adipocytes, followed by confocal microscopy to assess the degree of localization to the recycling endosome; FL (full-length Grp1-T280D), CC-Sec7 (Grp1 lacking the PH domain), Sec7-PH (Grp1-T280D lacking the CC domain); n = 10 cells per experiment with three independent experiments performed. (F) Recruitment of the Sec7-PH construct to the recycling endosome requires PI4KIIIα. The Sec7-PH construct was stably transfected into adipocytes that were treated with siRNA as indicated, followed by confocal microscopy to assess the degree of localization to the recycling endosome; SC (scrambled siRNA); n = 10 cells per experiment with three independent experiments performed. (G) Recruitment of the CC-Sec7 construct to the recycling endosome requires GRASP/IPCEF1. The CC-Sec7 construct was stably transfected into adipocytes that were treated with siRNA as indicated, followed by confocal microscopy to assess the degree of localization to the recycling endosome; n = 10 cells per experiment with three independent experiments performed.