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. 2018 Jan 22;8:1368. doi: 10.1038/s41598-018-19542-4

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© The Author(s) 2018

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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GGA2-depletion causes a drastic decrease in the EGFR protein expression. (a) Total lysates from ARPE-19 cells transfected with control (siCtrl), GGA1, GGA2, or GGA3 siRNAs were immunoblotted using indicated antibodies (Ab). Two sequences of siRNA (#1 and #2) were used for each GGA. Uncropped blots are presented in Supplementary Fig. 6. (b) Band intensities for EGFR were quantified in three or four experiments and mean values ± standard deviations (SD) were plotted. Differences between each GGA siRNA and siCtrl were analyzed by one-way ANOVA (P < 0.001) and Tukey’s honestly significant difference test (*P < 0.05, **P < 0.01, and ***P < 0.001). (c) Cells were treated with indicated siRNAs and were then fixed for immunofluorescence microscopy using anti-EGFR antibody. Nuclei were stained with Hoechst 33342 (blue); Bar, 20 μm. (d) GGA2-depleted cells re-expressing HA fused with siRNA-resistant full length GGA2 (HA-GGA2^kd-resi) were double immunolabeled with anti-HA and -EGFR antibodies. Signal intensity for EGFR was enhanced to detect differences. The arrow indicates a cell expressing HA-GGA2^kd-resi. Bar, 20 μm. (e) EGFR signal on the plasma membrane in HA-positive (n = 95) and -negative cells (n = 177) was quantified. Statistical analysis was performed using Student’s t-test (***P < 0.001). See Materials and Methods for details.