For SILAC, RAW264.7 cells expressing stably a FLAG-tagged form of MyD88-GyrB were cultured in arginine- and lysine-free RPMI 1640 (Invitrogen) supplemented with 10% dialyzed fetal bovine serum (Invitrogen), penicillin-streptomycin, and either l-arginine and l-lysine (light), l-arginine-HCl [13C6; CLM-2265 (R6)], and l-lysine-2HCl [4,4,5,5 D4; DLM-2640 (K4)] (medium), or l-arginine-HCl [13C6, 15N4; CLM-539 (R10)] and l-lysine-2HCl [13C6, 15N2; DLM-291 (K8)] (heavy) (Cambridge Isotope Laboratories) (75). For complete incorporation of labeled amino acids, cells were passaged three times in SILAC medium over a period of 5 days. The labeled cells were treated with 10 μM MPP for 20 min (heavy), followed by stimulation with 1 μM CpG-DNA for 60 min (medium and heavy). The medium was replaced by ice-cold phosphate-buffered saline (PBS), and cells were collected by cell scraping and centrifugation. Cell pellets were incubated with lysis buffer [LB; 20 mM Hepes/NaOH (pH 7.5), 1.5 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 10 mM β-glycerophosphate, 5 mM 4-nitrophenyl-phosphate, 10 mM sodium fluoride, and complete protease inhibitors (Roche)] supplemented with 0.5 % NP-40 for 20 min. Samples were cleared by centrifugation and loaded five times over M2 FLAG-bead–containing columns. Unbound proteins were removed by washing column with LB plus 0.1% NP-40, and proteins were eluted at pH 3.5 in water supplemented with 100 mM glycine, 50 mM NaCl, 0.1% NP-40, and Roche complete protease inhibitors. The proteins were concentrated by trichloroacetic acid precipitation and dissolved in SDS–polyacrylamide gel electrophoresis (PAGE) loading buffer (Bio-Rad). The dissolved proteins were combined, followed by separation on a 10 % Bis-Tris gel (Bio-Rad) and staining with SYPRO Ruby protein stain (Invitrogen). The entire lane was cut into individual bands and analyzed by LC-MS/MS using a nanoACQUITY ultra-performance liquid chromatography (UPLC) (Waters) coupled to an Orbitrap Elite high-resolution mass spectrometer (Thermo Fisher Scientific).

The protein gel bands were reduced with dithiothreitol and alkylated with iodoacetamide and then digested overnight with trypsin (Promega). The digest was introduced into the instrument by on-line chromatography using reversed-phase (C18) ultrahigh-pressure LC on a nanoACQUITY UPLC (Waters). The column used was a Waters BEHC18 with an inner diameter of 75 μm and a bed length of 10 cm. The particle size was 1.7 μm. Tryptic peptides were gradient-eluted over a gradient [0 to 70% B for 60 min and 70 to 100% B for 10 min, where B was 70% (v/v) acetonitrile, 0.2% formic acid] using a flow rate of 250 nl/min into the high-resolution Orbitrap Elite through a noncoated spray needle with voltage applied to the liquid junction.

Data-dependent scanning was incorporated to select the 20 most abundant ions (one microscan per spectrum; precursor isolation width, 2.0 Da; 35% collision energy; 10-ms ion activation; 15-s dynamic exclusion duration; 5-s repeat duration; and a repeat count of 1 from a full-scan mass spectrum at 60,000 resolution for fragmentation by collision-activated dissociation). Database searches were performed using raw files in combination with Andromeda search engine that is part of the MaxQuant software (version developed at the Max Planck Institute (76). The SwissProt 2012_08 (537,505 sequences; 190,795,142 residues) (taxonomy: Mus musculus (16,605 sequences) database was used for peptide and protein identification. MaxQuant was also used to quantitate peptides and proteins and to provide ratios generated in Excel format. Protein assignments were made on the basis of both MS and MS/MS spectra, whereas peptide quantitation was based solely on MS data. The following residue modifications were allowed in the search: carbamidomethylation on cysteine (fixed modification), oxidation on methionine (variable modification), label:13C(6) on arginine, label:13C(10) on arginine, label:13C(4) on lysine, and label:13C(8) on lysine. The MS1 mass tolerance was set to 15 parts per million, the MS/MS tolerance was set to 0.5 Da, and protein false discovery rate (FDR) was set to 0.01. The identifications from the automated search were verified by manual inspection of the raw data.

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