For affinity purification (AP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, clarified whole-cell lysates (10 mg protein per sample) were extracted with immobilized anti-HA beads (Pierce HA-Tag IP/Co-IP kit, Thermo-Fisher Scientific, PI23610). After a 3-h incubation, the beads were washed three times with lysis buffer and twice with HPLC water. The proteins bound on beads were eluted with 150 µl of 0.15% trifluoroacetic acid (TFA), neutralized with 100 mM NH4HCO3, and digested with trypsin. The tryptic peptides were purified by 200 µl C18 stage tips (Thermo Scientific, Rockford, IL, USA) and analyzed by Q-Exactive LC-MS/MS69. The tryptic peptides from anti-HA IP complexes were separated on a 50-cm Easy-Spray column with a 75-µm inner diameter packed with 2 µm C18 resin (Thermo Scientific, Odense, Denmark). The peptides were eluted over 120 min (250 nl/min) using a 0 to 40% acetonitrile gradient in 0.1% formic acid with an EASY nLC 1000 chromatography system operating at 50 °C (Thermo-Fisher Scientific). The LC was coupled to a Q-Exactive mass spectrometer70 by using a nano-ESI source (Thermo-Fisher Scientific). Mass spectra were acquired in a data-dependent mode with an automatic switch between a full scan and up to 10 data-dependent MS/MS scans. Target value for the full scan MS spectra was 1e6 with a maximum injection time of 120 ms and a resolution of 70,000 at m/z 400. The ion target value for MS/MS was set to 1,000,000 with a maximum injection time of 120 ms and a resolution of 17,500 at m/z 400. The first mass for the MS/MS was set to 140 m/z and the normalized collision energy was set to 27. Unassigned, as well as charge states 1 and >5 were ignored for MS/MS selection. Repeat sequencing of peptides was kept to a minimum by dynamic exclusion of sequenced peptides for 12 s71.