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. 2022 Mar 15;18(11):2656–2670. doi: 10.1080/15548627.2022.2046449

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© 2022 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 5. — PA attenuates the interaction of ATG3-ATG8e or ATG6-VPS34 by binding to GAPCs or PGK3. (A and B) GST affinity‐isolation assay showed the suppressed ATG3-ATG8e association by GAPCs (A, PGK3-His was used as a negative control) or ATG6-VPS34 association by PGK3 (B, GAPC1-His was used as a negative control). The pellet fraction was eluted and analyzed by immunoblotting using anti-His antibodies (upper panel). Input of GAPCs-His or PGK3-His (middle panel) and equal aliquots of glutathione beads loaded with GST-ATG3 or GST-ATG6 or GST (separated by SDS-PAGE and stained with Coomassie Brilliant Blue, bottom panel) were shown. The experiments were biologically repeated for three times and representative images were shown. (C) GAPCs-Flag and PA inhibit the interaction of ATG3-mCherry and GFP-ATG8e in vivo. GAPCs-Flag or GFP were transiently co-expressed in N. benthamiana leaves with ATG3-mCherry and GFP-ATG8e. After immunoprecipitated by GFP affinity magnetic beads, ATG3-mCherry, GFP-ATG8e, and GAPCs-Flag were detected by immunoblot analyses with anti-mCherry, anti-GFP, and anti-Flag antibody, respectively. (D) PGK3-Flag and PA inhibit the interaction of VPS34-mCherry and ATG6-GFP in vivo. PGK3-Flag or GFP were transiently expressed in N. benthamiana leaves with VPS34-mCherry and ATG6-GFP. After immunoprecipitated by GFP affinity magnetic beads, VPS34-mCherry, ATG6-GFP, and PGK3-Flag were detected by immunoblot analyses with anti-mCherry, anti-GFP, and anti-Flag antibody, respectively.