Figure 6.
Quantitative phosphoproteomics pipeline identifies the mitotic downstream substrates of TBK1. (A) Workflow for TMTpro-based phosphoproteomics of ∼20 M (million) mitotic cells. 16plex proteomics was performed in quadruplicate on samples harvested 1 h after mitotic release. (B) Volcano plots (log10 [P value] versus log2 ratio) for representing phosphorylation sites that are affected by the loss of TBK1. Proteins are shown in black circles (non-significant) and red circles (Tier 1 significant). The circles were color-coded for the motif that most likely fit TBK1 (blue). Bolded black circles categorize known mitotic proteins (GO:0000278). The inset indicated additional color coding for the statistical analysis. (C) Volcano plot scoring each identified substrate against the 303 kinase motifs panel from Johnson et al. (2023) to determine statistically significant enriched activated kinases. (D) Motif analysis using the pLogo tool identifies the motif for the substrate candidates based on synthetic peptide analysis. The y-axis is the log odds of the binomial probability. (E) Gene Ontology terms enrichment analysis among the Biological Processes for enriched phosphosites Tier 2. Mitotic and cell cycle–related classes were significantly enriched (left panel). Enrichment map networks, each node represents a gene set (i.e., a GO term) and each edge represents the overlap between two gene sets (right panel). (F and G) Representative Western blot and semiquantitative analysis of pAurora A and B/Aurora A and B normalized to vinculin in asynchronous and mitotic HeLa and TBK1 KO cells. RO-3306 was used for synchronization. Error bars indicate ± SEM; n = 3 independent experiments. (H and I) Representative Western blot and semiquantitative analysis of pAurora A and B/Aurora A and B normalized to vinculin in asynchronous and mitotic FKBP12F36V-NAP1 DLD-1 cells. dTAGv-1 treated cells occurred 2 h prior to and during G2 release. RO-3306 was used for synchronization. Error bars indicate ± SEM; n = 3 independent experiments. One dot = one independent experiment. Student’s t test was performed for WB statistical analysis. *P < 0.05, **P < 0.01, ns = not significant. Source data are available for this figure: SourceData F6.

Quantitative phosphoproteomics pipeline identifies the mitotic downstream substrates of TBK1. (A) Workflow for TMTpro-based phosphoproteomics of ∼20 M (million) mitotic cells. 16plex proteomics was performed in quadruplicate on samples harvested 1 h after mitotic release. (B) Volcano plots (log10 [P value] versus log2 ratio) for representing phosphorylation sites that are affected by the loss of TBK1. Proteins are shown in black circles (non-significant) and red circles (Tier 1 significant). The circles were color-coded for the motif that most likely fit TBK1 (blue). Bolded black circles categorize known mitotic proteins (GO:0000278). The inset indicated additional color coding for the statistical analysis. (C) Volcano plot scoring each identified substrate against the 303 kinase motifs panel from Johnson et al. (2023) to determine statistically significant enriched activated kinases. (D) Motif analysis using the pLogo tool identifies the motif for the substrate candidates based on synthetic peptide analysis. The y-axis is the log odds of the binomial probability. (E) Gene Ontology terms enrichment analysis among the Biological Processes for enriched phosphosites Tier 2. Mitotic and cell cycle–related classes were significantly enriched (left panel). Enrichment map networks, each node represents a gene set (i.e., a GO term) and each edge represents the overlap between two gene sets (right panel). (F and G) Representative Western blot and semiquantitative analysis of pAurora A and B/Aurora A and B normalized to vinculin in asynchronous and mitotic HeLa and TBK1 KO cells. RO-3306 was used for synchronization. Error bars indicate ± SEM; n = 3 independent experiments. (H and I) Representative Western blot and semiquantitative analysis of pAurora A and B/Aurora A and B normalized to vinculin in asynchronous and mitotic FKBP12F36V-NAP1 DLD-1 cells. dTAGv-1 treated cells occurred 2 h prior to and during G2 release. RO-3306 was used for synchronization. Error bars indicate ± SEM; n = 3 independent experiments. One dot = one independent experiment. Student’s t test was performed for WB statistical analysis. *P < 0.05, **P < 0.01, ns = not significant. Source data are available for this figure: SourceData F6.

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