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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 3. — GAPCs interacted with ATG3, and PGK3 interacted with ATG6, at endoplasmic reticulum (ER). (A) Liposome binding assay showed the correlation between bound GAPCs or PGK3 and PA. Purified His-tagged GAPCs or PGK3 were incubated with liposomes containing di18:1-PC only or di18:1-PA/PC (1/3 mole ratio). 1×, 4×, and 10× refer to the concentration gradient of PC or PA/PC liposomes used. Bound (pellet with liposomes, P) GAPCs or PGK3 and non-binding protein (supernatant with liposomes, S) were detected by immunoblotting. (B) GST pull-down assays revealed that GAPC1-His and GAPC2-His directly interact with GST-ATG3. Recombinantly expressed (from E. coli) GST or GST-ATG3 proteins were incubated with GAPC1-His or GAPC2-His and analyzed by immunoblot assays using an anti-His antibody (upper panel). Input of His-tagged proteins (middle panel) and equal aliquots of glutathione beads loaded with GST-ATG3 or GST (separated by SDS-PAGE and stained with Coomassie blue, bottom panel) were shown. (C) Co-IP assay demonstrated the interaction between ATG3-mCherry and GAPC1-GFP or GAPC2-GFP in vivo. GFP-tagged GAPCs was transiently co-expressed with mCherry-tagged ATG3 in N. benthamiana leaves and immunoprecipitated by GFP affinity magnetic beads. Resultant immunoprecipitation (IP) and cell lysate were analyzed by immunoblotting using anti-GFP or anti-mCherry antibodies. (D) Fluorescence observation of N. benthamiana leaf epidermal cells co-expressing GAPC1-cYFP/GAPC2-cYFP and nYFP-ATG3 with ER marker HDEL-mCherry. Representative images were shown (bar = 50 μm). (E) GST pull-down assays revealed that PGK3-His directly interacts with GST-ATG6. Recombinantly expressed (from E. coli) GST or GST-ATG6 proteins were incubated with PGK3-His and analyzed by immunoblot assays using an anti-His antibody (upper panel). Input of His-tagged proteins (middle panel) and equal aliquots of glutathione beads loaded with GST-ATG6 or GST (separated by SDS-PAGE and stained with Coomassie blue, bottom panel) were shown. (F) Co-IP assay demonstrated the interaction between ATG6-mCherry and PGK3-GFP in vivo. GFP-tagged PGK3 was transiently co-expressed with mCherry-tagged ATG6 in N. benthamiana leaves and immunoprecipitated by GFP affinity magnetic beads. Resultant immunoprecipitation (IP) and cell lysate were analyzed by immunoblotting using anti-GFP or anti-mCherry antibodies. (G) Fluorescence observation of N. benthamiana leaf epidermal cells co-expressing PGK3-cYFP and nYFP-ATG6 with ER marker HDEL-mCherry. Representative images were shown (bar = 50 μm).