Fig. 5: Global protein phosphorylation changes after PP2A-inhibition in directSLiM^LIE9 cells.

A Experimental design: mitotically arrested directSLiM^LIE9 and directSLiM^AAA cells were treated with rapamycin, rapalog or vehicle before harvest (n = 3 for each condition). Following cell lysis, proteins were digested, and peptides were TMT-labeled. Next, phosphopeptides were enriched with Fe-NTA spin column, followed by high-pH reversed-phase fractionation and measurement by LC-MS. B Zoomed-in right upper quadrant of volcano plot showing upregulated phosphorylation sites after treatment with rapamycin or rapalog. Solid dots are either validated substrates, predicted substrates or interactors of validated PP2A-B56 substrates (see methods for details). Only hits above a -Log₁₀(P value = 0.05) (Two- sided Student’s t-test) and >1.5-fold change are color coded. C Heat map of phosphorylation sites upregulated after PP2A-B56 inhibition in directSLiM^LIE9 cells. Each row represents the intensity of a phosphorylation site (n = 149). Intensities are normalised to vehicle treated condition for each cell line to show the relative fold-change upon rapamycin/rapalog addition. Gene names are added for all phospho-sites from validated substrates or interactors of validates substrates. D Percentage of Aurora kinase, PLK1 and CDK consensus motifs in the increasing and non-changing phosphorylation sites. E Sequence logo of non-proline directed upregulated phosphorylation sites normalized against background (all non-changing phosphosites used as background). F Heat map of phosphorylation sites upregulated after PP2A-B56 inhibition in directSLiM^LIE9 cells. Each row represents the z-scored intensity of a phosphorylation site (n = 155). The label (ii) refers to phospho-sites in doubly phosphorylated peptides. Source data are provided as a Source Data file.