Title Loaded From File

Ormed as described in `Materials and methods’. We reproducibly identified 6956 phosphorylation websites on 1850 proteins with single amino acid accuracy (in accordance with the PTM score; Olsen et al, 2006), more than 60 of which had been novel with respect for the phosphorylation web site database Expasy (containing all Swiss-Prot/TrEMBL entries; http:// expasy.ch) in addition to a recent phosphoproteome study in the mouse liver cell line Hepa1-6 (Pan et al, 2008) (Supplementary Table S1). The overlap among our two completely independent experiments was 639 , based on the experiment referred to (Figure 2A). For bioinformatic analyses, we focused on reproducibly identified phosphorylation websites, if not indicated otherwise. Validation of phosphosites identified by mass spectrometry could be completed by immunoblotting in instances exactly where phosphorylation site-specific antibodies are available. We confirmed the regulated phosphorylation of GSK3b at S9 and ribosomal protein S6 at S235/236 (Supplementary Figure S2), the phosphorylation of p38 MAPK (Mapk14) at T180 and Y182 (Supplementary Figure S1) and of ERK1 MAPK (Mapk3) 2010 EMBO and Macmillan Publishers LimitedPhosphoproteome of TLR-activated macrophages G Weintz et alAArg `0′ Lys `0’Arg `6′ Lys `4’Arg `10′ Lys `8’SILAC PoolWT unstim.KO 15 minWT 15 min PoolWT unstim.KO 4 hWT 4 h PoolWT unstim.KO unstim. SILAC medium Day 0 1 BM Depl. of adherent cellsStimulation, pool cell lysates Soluble fraction Digest SCX TiO2 Res. chromatin pellet SDS AGE Digest TiOBExpansion IL-3, IL-6, SCF M-CSFIdentification and quantification of phosphopeptides by LC S/MSRel. abundance13 Peptide 1 Peptide 2 WT unstim. KO (un)stim. WT stim. 16 17 Differentiation M-CSF Stimulation and lysism/zCMio cells80 70 60 50 40 30 20 ten 0 1 3 5 7 9 11 13 15 17 Days in cultureDAVFPSIVGRPRLabelling efficiency 905Figure 1 Experimental program and style. (A) Technique for international and quantitative evaluation of LPS-induced phosphorylation. Bone marrow cells from wild form (WT) and Dusp1-deficient (KO) mice have been SILAC encoded with regular and stable isotope-substituted arginine and lysine amino acids, making three states distinguishable by mass ((m/z) mass/charge). Every population was stimulated with LPS for 15 min or four h or left un-treated. Unstimulated wild-type cells were included in all 3 pools as a common reference point. Cell lysates to be directly compared were pooled, fractionated and enzymatically digested into peptides, and phosphopeptides were enriched on TiO2 beads and analysed by online LC-MS/MS. Owing towards the mass shifts introduced by the SILAC amino acids mass spectra of labelled peptides Capsid Inhibitors Related Products revealed SILAC triplets (same peptide in the 3 cell populations), using the intensities of the peaks reflecting the relative amounts of a peptide within the three conditions. This SILACbased method permitted high-accuracy quantification of phosphopeptides and, in most instances, localisation from the phosphate group with single amino acid accuracy. Two independent experiments were performed. (B) Optimised protocol for SILAC of bone marrow-derived macrophages. (C) Cell proliferation under the SILAC protocol. Total quantity of cells at unique time points through SILAC labelling (mean tandard deviation from two independent experiments). (D) Labelling efficiency. Representative peptide containing two arginine residues. The arrow indicates the position of partially labelled peptide.at T203 and Y205 (4D). Also, the powerful phosphorylation of ATF2 and TTP (Zfp36) at vari.