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. 2019 Jul 9;10:3020. doi: 10.1038/s41467-019-10865-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — US11 expression retains FcRn in the endoplasmic reticulum (ER). a, c FcRn or CD71 appearance in the endosome in HeLa^FcRn+US11 and HeLa^FcRn cells. HeLa^FcRn+US11 and HeLa^FcRn cells were transfected with a plasmid expressing human CD71-GFP (green). Both cells were immunostained for FcRn (green) and EEA1 (red). b, d Averages of the EEA1 and FcRn or CD71 co-localization in HeLa^FcRn+US11 and HeLa^FcRn cells. Pearson’s correlation coefficient was measured in ten different optical regions (100 cells total) in each experiment. Error bars represented mean ± SEM of ten different optical regions. The nuclei were stained with DAPI (blue); co-localization of two molecules appears in yellow. Scale bar: 10 μm. Similar images were seen from at least three independent staining experiments. EEA1: early endosome antigen 1. ***P < 0.001, NS: no significance by Student’s t-test. e, f β₂m or US11 does not co-immunoprecipitate with US11 or β₂m protein. Cell lysates from HeLa^FcRn+US11 (lane 1), HeLa^FcRn (lane 2), HeLa^US11 (lane 3), and HeLa cells (lane 4) were immunoprecipitated by anti-HA mAb (e), anti-β₂m Ab (f). All immunoprecipitates or cell lysates (20 μg) as internal controls were subject to western blotting with corresponding Abs as indicated. g, h Sensitivity of US11-associated FcRn HC to Endo-H digestion. g Native FcRn in cell lysates (top panel) or proteins immunoprecipitated by HA mAb (bottom panel) were digested by mock (lanes 1 and 4), Endo-H (lanes 2 and 5), or PNGase F (lanes 3 and 6) for 2 h at 37 °C, respectively. All immunoprecipitates or cell lysates (20 μg) were subject to western blotting with corresponding Abs as indicated. The ratio of Endo H-resistant (Endo-H^R) FcRn HC to Endo H-sensitive (Endo-H^S) FcRn HC from HeLa^FcRn+US11 and HeLa^FcRn cells were compared by the ratio of the band density of glycosylated FcRn to that of the deglycosylated FcRn (h). The band density of Endo-H-sensitive or resistant FcRn (g, top panel, lanes 2 and 5) was quantified by the software Image Lab 5.2. *P < 0.05 by Student’s t-test. Error bars represented mean ± SEM of three independent repeats. R: Resistant; S: Sensitive