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. 2021 Oct 17;18(6):1350–1366. doi: 10.1080/15548627.2021.1976965

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© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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Figure 7. — The AIM-like motif of SH3P2 is dispensable for its trafficking in endocytosis. (A) Yeast-two-hybrid assay for analyzing the interaction between SH3P2 and Auxilin2. (B) Immunoprecipitation assay showed Auxilin2-3HA is associated with both SH3P2-GFP and SH3P2^Y325A,V328A-YFP. Auxilin2-3HA was co-expressed with SH3P2-GFP and SH3P2^Y325A,V328A-YFP in Arabidopsis protoplasts respectively. Lysates were immunoprecipitated by GFP-Trap and detected by anti-GFP or anti-HA antibodies. (C) Subcellular analysis showed that Auxilin2-YFP is colocalized with both SH3P2-RFP and SH3P2^Y325A,V328A-RFP in Arabidopsis protoplasts. Similar results were obtained from three different independent experiments. The right column shows the scatterplot images obtained from ImageJ with the PSC colocalization plug-in. The linear Pearson correlation coefficient (rp) and the nonlinear Spearman correlation coefficient (rs) indicate the extent of colocalization with the value of +1.0 for complete colocalization. (D) Both SH3P2-GFP and SH3P2^Y325A,V328A-GFP signals were mainly detected in the cytosol and on the plasma membrane, as well as the cell pate forming sites (arrows) in Arabidopsis root cells. (E) Both SH3P2-GFP and SH3P2^Y325A,V328A-GFP were associated with the CCV (clathrin-coated vesicle) marker CLC2-RFP in transgenic plants. (F-G) A treatment with FM 4–64 dye or cotreatment with brefeldin A (BFA) showed that uptake of FM 4–64 and overaccumulation of FM 4–64 dye-labeled BFA-bodies were similar in SH3P2-GFP and SH3P2^Y325A,V328A-GFP transgenic plants. Consistent results were obtained from three independent experiments.