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. 2020 Feb 14;10:2658. doi: 10.1038/s41598-020-59537-8

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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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Regulation of phosphorylations by UGS and its components. (a) The phosphorylation status of signaling proteins was measured using antibody array technology from stimulated neurovascular cells. Protein phosphorylation levels were compared with those of control samples to obtain phosphorylation ratios. A phosphorylation profile was obtained by hierarchically clustering approximately 310 proteins showing significant variation (standard deviation > 1.0) over all neurovascular cell types. The red and green colors reflect high and low protein phosphorylation, respectively, as indicated by the scale bar. A detailed image is shown in Supplementary Fig. S9. (b) Enriched signaling pathways from protein phosphorylation in stimulated neurovascular cells were analyzed with the Signaling Pathway Impact Analysis (SPIA) program. The horizontal axis represents pathway overrepresentation (P_NDE), while the vertical axis indicates pathway perturbation (P_PERT). The red and blue circles on the right of the red oblique lines represent significant pathways after FDR correction (0.01 and 0.05, respectively) of the global p-values (P_G), representing the pathway ranks calculated from the combination of both P_NDE and P_PERT. The list of phospho-based pathways is shown in Supplementary Table S2. (c) The distribution of significant pathways (FDR < 0.01) from SPIA was displayed in connection with neurovascular cell types. A detailed image is shown in Supplementary Fig. S10. (d) From the phosphorylation profile, differentially phosphorylated proteins were defined as proteins showing a change of phosphorylation status above 2 or below 0.5 compared with control samples in each stimulated neurovascular cell type. Then, the effects of each herbal component were measured by observing recovery of phosphorylation. Recovered proteins were defined as those whose phosphorylation was restored to normal levels (phosphorylation ratio between 0.5 and 2). Based on the recovery pattern of phosphorylation, we measured the number of proteins recovered in common between two herbal components. After extending this measurement to all combinations of herbal components, we constructed the therapeutic network in terms of commonly recovered phosphoproteins. Then, the correlation between neurovascular cell types was assessed in terms of the similarity of patterns of phosphorylation recovery by herbal components.