BMDMs (108 cells per condition) were lysed with 1 ml of radioimmunoprecipitation assay buffer [150 mM NaCl, 50 mM tris (pH 7.4), 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.25% sodium deoxycholate, and protease and phosphatase inhibitors]. Immunoprecipitation was performed by incubation of lysates with 2 μg of anti-BCAP antibody (4L8E6) (4) overnight (4°C), followed by subsequent enrichment using protein A agarose beads (Calbiochem). The resulting protein complexes were washed several times in lysis buffer and twice in 50 mM ammonium bicarbonate buffer and were subsequently reduced, alkylated, and digested into peptides using trypsin (V5280, Promega). Peptides were loaded onto a 3-cm self-packed C18 capillary precolumn (5 μM; Reprosil, Dr. Maisch). After a 10-min rinse (0.1% formic acid), the precolumn was connected to a 25-cm self-packed C18 (3 μM; Reliasil, Orochem) analytical capillary column (inner diameter, 50 μm; outer diameter, 360 μm) with an integrated electrospray tip (∼1-μm orifice). Online peptide separation followed by MS analyses was performed on a two-dimensional nano–liquid chromatography system (nanoACQUITY UPLC system, Waters Corporation). Peptides were eluted using a 150-min gradient with solvents A [H2O/formic acid, 99.9:1 (v/v)] and B [acetonitrile/formic acid, 99.9:1 (v/v)]: 10 min from 0 to 10% B, 105 min from 10 to 40% B, 15 min from 40 to 80% B, and 20 min with 100% A. Eluted peptides were directly electrosprayed into an Orbitrap Q Exactive MS (Thermo Fisher Scientific) equipped with a high-energy collision cell [high-energy C-trap dissociation (HCD)].

The MS was operated in a data-dependent mode to automatically switch between MS and MS/MS acquisitions. Each full scan [mass/charge ratio (m/z) from 300 to 1500] was acquired in the Orbitrap analyzer (resolution, 70,000), followed by MS/MS analyses on the top 20 most intense precursor ions that had charge states greater than one. The HCD MS/MS scans were acquired using the Orbitrap system (resolution, 17,500) at a normalized collision energy of 28%. The precursor isolation width was set at 2 m/z for each MS/MS scan, and the maximum ion accumulation times were as follows: MS (100 ms) and MS/MS (100 ms). MS/MS data files were searched using the CoMEt algorithm (48), and the data were further processed using the Institute for Systems Biology’s Trans Proteomic Pipeline (49). Static modification of cysteine (carbamidomethylation; 57.02 Da) was used in the search. For precursor area quantification of LRRFIP2, PIK3AP1, and FLII peptides, the data files were imported into the Skyline software package (50), whereupon the retention time windows for each peptide was determined on the basis of coelution the MS/MS identification. For each peptide, the relative intensity of the precursor ions was quantified using this retention time window.

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