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. 2020 Jul 10;3:368. doi: 10.1038/s42003-020-1063-5

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© The Author(s) 2020

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. 1 — a Schematic representation of acyl-biotin exchange (ABE) protocol coupled with SILAC for quantitation of palmitoylated proteins via LC-MS/MS. Briefly, Jurkat cells grown in heavy SILAC media (blue) were lysed and free thiols were blocked with NEM. After cleaving palmitoyl groups with HA, previously palmitoylated thiols are biotinylated with HPDP-biotin, allowing for streptavidin enrichment. For control cells grown in light SILAC media (red), HA is omitted, preventing biotinylation. Proteins quantified with heavy/light intensity ratios >2.0 are considered enriched and palmitoylated. b Volcano plot showing high-confidence palmitoylated proteins prior to stimulation (n = 6, p < 0.05). Proteins found in five or more other palmitome studies are highlighted in orange. c Volcano plot showing high-confidence palmitoylated proteins 10 min after anti-CD28/anti-CD3 costimulation (n = 4, p < 0.05). d Comparison of high-confidence palmitoylated pools before and after stimulation. Only proteins from b and c that displayed a p-value < 0.05 are considered. These p-values and additional statistical parameters are detailed in Supplementary Data 1. e Analysis of palmitoylated proteins compared to unenriched (background) proteins, with three metrics: at least five references in previous palmitome studies, at least one predicted palmitoylation site (CSSPalm 4.0, high cutoff), and at least one predicted transmembrane domain (TMHMM 2.0). f STRING functional analysis of proteins enriched for palmitoylation 10 min after TCR stimulation. A clear cluster of Gα and Ras-related proteins is observed. Several interesting candidates are highlighted, including VAMP7 and DHHC18, which were investigated in more detail in this study. g Gene Ontology (GO) term enrichment of stimulation-dependent palmitoylation candidates, generated using BiNGO. Cluster size indicates the number of genes in each node, while color indicates statistical significance of enrichment.