br Materials and methods br Acknowledgments

Materials and methods
Acknowledgments We thank the Institut National de Recherche en Sciences et Technologies pour l׳Environnement et l׳Agriculture (France), the Commissariat à l׳Energie Atomique et aux Energies Alternatives (France) through the Transversal Toxicology Program (Pptox), the Agence Nationale de la Recherche program “ProteoGam” (ANR-14-CE21-0006-02) and the Agence Nationale de la Recherche CESA program “GAMMA” 021 02 “Variability-Adaptation-Diversity and Ecotoxicology in Gammarids” (2012-2015) for financial support. The authors thank Michalis Averof (IGFL, Lyon) for his kind gift of P. hawaiensis animals and Bernard Clément (ENTPE, Vaux en velin) for generously supplying H. azteca animals.
[please fill in right-hand column of the table below]
Experimental Design, Materials and Methods
Acknowledgements This research was funded by the NSF (1149387) (CP).
Experimental design, materials and methods
Direct link to deposited data Data is available through the PRIDE proteomics database through the following link http://www.ebi.ac.uk/pride/archive/projects/PXD001272 and will also be made available through the giardiadb.org website later in 2015.
Conflict of interest
Acknowledgements SJE acknowledges funding from the Australian Government in the form of an APA scholarship, as well as financial support from Macquarie University. SJE wishes to thank Dr Jacqui Upcroft for supplying the Giardia samples used and for the ongoing support received from colleagues at Microbial Screening Technologies. PAH wishes to thank Justin Lane for continued support and encouragement.
Specifications Table Value of the data Data Phosphorylation is a dynamic modification, and therefore to fully understand the meaning of a specific phosphorylation, its MC1568 cost must be known. The stability is an output of the activity of the regulatory kinase and phosphatase (Fig. 1A). In order to understand the dynamic nature of phosphorylation sites, we took advantage of the fact that during mitosis over 75% of the human proteome (>7000 proteins) is phosphorylated, with those proteins phosphorylated on the majority of all potential phosphorylation sites [2]. As cells exit mitosis these phosphorylations are removed in a highly organized, sequential manner [3]. Therefore, mitotic exit provides an excellent experimental system to rapidly analyze the temporal dynamics of phosphorylation. We recently performed a global phosphoproteomics analysis comparing mitosis to early mitotic exit [1], and here we present detailed methods and additional data from this study. This additional information can be used by the wider research community to infer a potential function of a phosphorylation sites based on our reported mitotic temporal dynamics, or as predictive tool for the stability of a novel phosphorylation based amino acids surrounding the phosphosite.
Experimental design, materials, and methods
SILAC labeling HeLa cells were SILAC-labeled by culturing in DMEM where the natural “light” Lysine and Arginine were replaced by “heavy” isotope-labeled amino acids 13C615N4-l-Arginine (Arg 10) and 13C615N2-l-Lysine (Lys 8) (Silantes GmBH), which was supplemented with 10% dialyzed FBS and 4mM glutamine. To ensure complete labeling of >97%, cells were cultured for approximately six doublings in heavy or light media, with fresh media replaced every two days and sub-culturing performed when cells reached 90% confluence. After labeling, cells were synchronized as per Fig. 1B. Mitotic cells were enriched by shake off, and both light and heavy labeled samples were treated with 25µM MG132 for 15min. Heavy labeled samples were then treated with 10µM RO3306 (RO) for a further 15min, with both samples then harvested by centrifugation at 4°C (Fig. 1C). Three biological replicates were prepared, and in one replicate, the heavy/light labels were switched to provide an internal labeling control.