TAP-MS was performed as previously described (Jung et al., 2018). Cells were collected in 15 mL ice-cold NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 0.5 mM EDTA, and 0.5% NP-40; freshly supplemented with proteinase and phosphatase inhibitors) and shaken at 4 °C for 20 min. Lysates were subjected to centrifugation at 4 °C and 13,148 g for 15 min. Transferred supernatants were incubated with streptavidin-conjugated beads (Amersham) for 1 h at 4 °C. After three washes with NETN buffer, the beads were transferred to a new tube, and interacting proteins were eluted with 1.5 mL NETN buffer and 2 mg/mL biotin (Sigma) for 90 min at 4 °C. The eluted proteins were transferred and incubated with S-protein beads (Novagen) for 1 h. The beads were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) after three washing steps. Protein bands were excised and subjected to mass spectrometry analysis. After excised gel bands were cut into ∼1 mm3 pieces, in-gel trypsin digestion was performed. Dried samples were reconstituted in 5 μL of high-performance liquid chromatography (HPLC) solvent A (2.5% acetonitrile, 0.1% formic acid). By packing 5-μm C18 spherical silica beads into a fused-silica capillary (100-μm inner diameter × ∼20-cm length) with a flame-drawn tip, a nanoscale reverse-phase HPLC capillary column was created. After the column was equilibrated, each sample was loaded onto the column using a Famos autosampler (LC Packings), and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). Eluted peptides were subjected to electrospray ionization and then entered into an LTQ Velos ion-trap mass spectrometer (Thermo). After peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide, peptide sequences were determined by matching protein databases (the human IPI database version 3.6) with the acquired fragmentation patterns using the software program SEQUEST (version 28) (Thermo). The specificity of the enzyme was set to partially tryptic with two missed cleavages. Carboxyamidomethyl (cysteines, fixed) and oxidation (methionine, variable) were included in the modification. Mass tolerance was set to 2.0 for precursor ions and 1.0 for fragment ions. To achieve a false discovery rate of less than 1% at the peptide level, spectral matches were filtered based on the target-decoy method. Finally, only tryptic matches were reported, and spectral matches were manually examined. Peptides that matched to multiple proteins were assigned so that only the most logical protein was included (Occam’s razor).

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