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. 2021 Mar 25;12:1863. doi: 10.1038/s41467-021-22181-5

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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. 6 — a Principal component analysis of the phospho-proteomes of cells treated with 2i or Cdk8/19i at different time points. b Differentially regulated p-sites using a moderated t-test (limma, two-sided, FDR < 5%) at the indicated time points. The number of common p-sites between 2i and Cdk8/19i is shown together with the corresponding p.values calculated with a hypergeometric test (RF indicates “Representation factor”). c Gap statistics and K-means classified the regulated phospho-proteome into 14 different clusters. Centroids are indicated with a thick red (2i) or blue (Cdk8/19i) line. Only six clusters are shown in the figure. The other 8 clusters are shown in Supplementary Fig. 5C. Color gradient in line plots indicates proximity to the centroid of the cluster. d Changes in p-sites of kinases involved in mTOR signaling in 2i or Cdk8/19i (n = 4 independent cell lines). Statistically significant changes using a moderated t-test (FDR < 5%) relative to reference S/L are indicated with an asterisk (p-value < 0.05). e Changes in phosphorylation of S317 regulatory site of Chek1 as well as two known Chek1 substrates (n = 4 independent cell lines). Statistically significant changes using a moderated t-test (FDR < 5%) relative to reference S/L are indicated with an asterisk (p-value < 0.05). f Ranked p-sites by their absolute difference between 2i and Cdk8/19i. Mek2 p-sites identified in our dataset are indicated. g Phosphorylation changes of Mek/Erk regulatory sites. Points indicate the average of n = 4 independent cell lines. Statistically significant changes using a moderated t-test (FDR < 5%) relative to reference S/L are indicated with an asterisk (p-value < 0.05). h Schematic model of Myc phospho-regulation in S/L and 2i. Changes in the T58/S62 di-phosphorylated Myc peptide and the S62 monophosphorylated are shown (n = 4 independent cell lines). Statistically significant changes using a moderated t-test (FDR < 5%) relative to reference S/L are indicated with an asterisk (p-value < 0.05). Source data for Fig. 6a are provided as Source Data file.