Lysates of cells infected with pHAGE virus expressing HA-tagged mortalin constructs were subject to immunoprecipitation using agarose beads conjugated with anti-HA antibody (Thermo Fisher Scientific) and, subsequently, to SDS-PAGE, as we previously described (22). Silver-stained gel bands were excised, destained, reduced, and tryptic-digested. Peptides were extracted from these gel pieces using trifluoroacetic acid and acetonitrile, dried under vacuum before MS analyses using an Eksigent nanoLC in line with an LTQ Orbitrap Velos (Thermo Fisher Scientific). Two technical replicate injections of 2 μl in succession were performed using instrument settings outlined in table S1. MS-grade solvents and reagents were used. MS data were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) according to details in table S1. All files for separate bands from a single lane (meaning the same sample) were searched together to produce a single output for each lane.

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