br Data analysis Annotation of upstream

Data analysis Annotation of upstream kinase was done using the reported minimal consensus motifs for each kinase [1] and using KinomeXplorer (http://kinomexplorer.info) [10]. This information is annotated in Supplementary Table S1. Simple data analysis of this table can be performed using Microsoft Excel and the filter function. Sequences for each kinase for the statistically significant dephosphorylated, and stable phosphosites were analyzed using motif enrichment analysis with Icelogo [11] and WebLogo 3 [12]. The results of this analysis are shown in Fig. 2A. Briefly, acidic residues (D, E) upstream (right) of the phosphorylation site are more commonly associated with stable phosphosites (Fig. 2A). Based on our simplistic model (Fig. 1A), these acidic residues could inhibit or reduce the preference of phosphatases for these phosphorylation sites, thereby creating a stable (long half-life) phosphorylation (Fig. 1A).
Quantitative immunofluorescence staining To validate the motifs observed in Fig. 2A, we performed quantitative immunofluorescence staining of cells undergoing phosphatase dependent mitotic exit (Fig. 2B). Cells, grown on Histogrip (Invitrogen) coated glass coverslips, were synchronized as per Fig. 1B, and harvested using ice cold 100% methanol at 0min (Metaphase), 15min (Early), 30min (Mid) 45min (Late) and 60min (Very Late) post addition of the Cdk1 inhibitor RO33306. Fixed cells were washed and blocked (3% BSA, 0.1% Tween 20 in PBS) for 30min, then incubated with primary SW033291 for pSerCdk (#2324, Cell Signaling Technologies) and β-tubulin [13] for 2h at room temperature in blocking solution. Mouse and Rabbit secondary Alexa 488 and 594 (Invitrogen) antibodies along with DAPI were used to visualize pSerCdk, microtubules, and DNA respectively. Images were captured using a Leica DM5500 microscope coupled with a Coolsnap HQ2 camera, using a Leica 100X or 40X APO 1.4 lens, powered by Leica LAS AF v3 software. To quantify pSerCdk levels in cells, a single in-focus plane was acquired using identical microscope settings for all conditions. Analysis was performed using Image J (v1.48, NIH) an outline drawn around each cell and circularity, area, mean fluorescence measured, along with several adjacent background readings. The Total Corrected Cellular Fluorescence (TCCF)=Integrated Density – (Area of selected cell×Mean fluorescence of background readings), was calculated. Box-plots and statistical analysis (ordinary one-way ANOVA, with Newman–Keuls correction for multiple comparisons) were performed using GraphPad Prism 6. For all β-tubulin and DAPI, 0.3µm z-sections were taken, de-convolved, and displayed as 2D maximum projections using Image J. False coloring and overlays were performed using Adobe Photoshop CC 2015 software.
Acknowledgments We thank Gillian Lehrbach (Tissue Culture), Will Hughes (Microscopy) Sean J. Humphrey, Daniel J. Fazakerley and Mark Cowley for assistance. We thank the PRIDE Team at the ProteomeXchange Consortium. This work was supported by the Cancer Institute NSW FRL fellowshipID#10/FRL/3-02, The Patricia Helen Guest Fellowship, and the Petre Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data, experimental design, materials and methods In the present work we provide the data generated to unveil the signaling pathways initiated by IL-2 and IL-15 in T-lymphocytes [1]. We include two tables containing quantitative and qualitative information about all the proteins and phosphotyrosine (pY)-containing peptides identified and quantified in the pY-immune complexes isolated from IL-2- and IL-15-treated T-lymphocytes, as well as the gene ontology analysis was performed. To assess the signaling networks initiated downstream of IL-2/IL-2R and IL-15/IL-15R complexes in T-lymphocytes, we followed the experimental workflow shown in Fig. 1. (A) Kit225 T-cells were grown in media containing either light (0/0), medium (6/4) or heavy (10/8) version of arginine and lysine until their proteome was completely labeled. Then, cells grown in light media were kept unstimulated and thus served as control whereas cells grown in medium and heavy media were stimulated with IL-2 and IL-15, respectively. After protein extraction, differentially labeled cell lysates were combined in 1:1:1 ratio. (B) Tyrosine phosphorylated proteins were enriched using phospho-specific antibodies and (C) fractionated on a SDS-PAGE. After protein in-gel digestion and peptide extraction, resulting peptides where either (D) enriched in phosphopeptides using TiO2 beads prior mass spectrometry (MS) analysis or (E) directly analyzed by a QExactive mass spectrometer. (F) Acquired raw mass spectra data were analyzed using the MaxQuant software and further data analysis was performed using David bioinformatics tools.