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. 2021 Nov 25;12:6877. doi: 10.1038/s41467-021-27279-4

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© The Author(s) 2021

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. 5 — a Co-immunoprecipitation assay showing the interaction between AGPAT2-GFP and HA-CDS1/2 in HEK296E cells. n = 3 biologically independent experiments. b Strep-tagged AGPAT2 and Flag-tagged CDS1 or CDS2 were expressed in HEK293F cells alone or together as indicated, followed by either a one-step affinity purification by anti-Flag or anti-Strep or a two-step affinity purification by anti-Flag and then anti-Strep. The Coomassie blue-stained gel shows purification of CDS1, CDS2 and AGPAT2, and co-purification of CDS1/2 and AGPAT2. n = 3 biologically independent experiments. c Co-immunoprecipitation assay showing the interaction between endogenous AGPAT2-sfGFP and CDS2-mScarlet in HeLa cells. AGPAT2 or CDS2 was tagged at their genomic locus with sfGFP or mScarlet by CRISPR. SKI: single sfGFP knockin at AGPAT2. DKI: mScarlet knockin at CDS2 and sfGFP knockin at AGPAT2. n = 3 biologically independent experiments. d Confocal imaging of fixed HeLa cells showing AGPAT2 and CDS2 tagged at their genomic loci with sfGFP and mScarlet, respectively. Cells were treated with high glucose DMEM (basal) or low glucose DMEM for 48 h or re-incubation with high glucose DMEM (Refed) for another 24 h. Bars = 10 μm. n = 3 biologically independent experiments. e Co-immunoprecipitation of mCherry-tagged AGPAT1, 2, 3 and HA-tagged CDS1 from transfected HEK293E lysates. n = 3 biologically independent experiments. f Co-immunoprecipitation of mCherry-tagged AGPAT2, 4, 5 and HA-tagged CDS1 from transfected HEK293E lysates. n = 3 biologically independent experiments. g Co-immunoprecipitation of mCherry-tagged AGPAT1, 2, 3 and HA-tagged CDS2 from transfected HEK293E lysates. n = 3 biologically independent experiments. h Co-immunoprecipitation of mCherry-tagged AGPAT2, 4, 5 and HA-tagged CDS2 from transfected HEK293E lysates. n = 3 biologically independent experiments.