Myc trap beads

Following mitochondrial isolation, mitochondria were gently lysed using 4.5 g glyco-diosgenin (GDN)/g protein in IP buffer [0.1 M Tris-HCl, 0.15 M NaCl, phospholipids (0.03 mg/ml phosphatidylethanolamine, 0.03 mg/ml phosphatidylglycerol, and 0.09 mg/ml phosphatidylcholine) and 1× cOmplete ULTRA Protease Inhibitor Cocktail]. Myc-tagged proteins were isolated using 10 µl Myc-Trap beads (per 10 cm dish; Chromotek). The supernatant (lysed mitochondrial sample) was incubated on a rotating wheel with beads overnight at 4°C. Subsequently, beads were washed in IP buffer. After washing, the supernatant was removed, and samples were analysed by western blotting.

All yeast strains were grown to log phase (2–3 × 10⁷ cells/ml). Afterwards, the cells were harvested and lysed in IP buffer (1 × PBS, 3 mM KCl, 2.5 mM MgCl₂, 0,5% Triton X-100 and protease inhibitors from Roche). 35 µl of this lysate was loaded onto an SDS-gel (lysate lanes). The supernatant was incubated for 1 h at 4 °C with GFP-Selector beads (NanoTag) (Fig. 5A,B,D, Supplementary Fig. S5.1A, 5.2A, 5.3B) or with Myc-trap beads (Chromotek) (Fig. 5C, Supplementary Fig. S5.3A). The beads were washed five times with IP buffer, and finally resuspended in 35 µl SDS-sample buffer. The entire eluate sample was loaded onto the SDS-gels.
Subsequently, the proteins were detected by Western blot analyses with the indicated antibodies (GFP (Chromotek) 1:4,000; c-myc (9E10) (Santa Cruz) 1:1,000; Hem15 and Grx4 each 1:5,000 and Aco1 1:2,000 (U. Mühlenhoff); Nop1 (Santa Cruz) 1:4,000; Hdf1 (Yku70) (Santa Cruz) 1:4,000). Signals were detected with the Fusion SL system (PeqLab) and FusionFX7 Edge (Fusion FX Vilber). To be able to detect several proteins in one experiment, the western blots were cut horizontally according to the size of the desired proteins to be able to detect each stripe with individual antibodies.

INS-1E cells were transfected at 60% confluence using Lipofectamine™ 3000 (ThermoFisher Scientific, Denmark) with plasmids coding for GFP-tagged GRP94, PI, ER-localized GFP [23 (link),24 (link)] or myc-tagged FKBP2, according to the manufacturer’s protocol. Proteins were immunoprecipitated (IP) using the magnetic GFP- or myc-trap beads (Chromotek, Germany). IP samples were reduced and alkylated, digested with trypsin/LysC and the resulting peptides were analyzed on a Bruker Impact II ESI-QTOF (Bruker Daltonics, USA) mass spectrometer (supplementary procedures).

For immunoprecipitation, cells transfected for 48 hr were lysed in the buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1 mM Na₃VO₄, 10 mM NaF), containing cOmplete Mini EDTA-free protease inhibitor cocktail (Roche). After centrifugation at 16,000 g, the supernatant was incubated with anti-FLAG agarose beads (Sigma) at 4°C for 2 hr or with anti-Myc antibodies (9E10) for 2 hr and Protein A Sepharose (GE Healthcare) at 4°C for 2 hr. Myc-trap beads (Chromotek) were used to pull-down Par3-containing protein complexes. The beads were washed three times in lysis buffer, and subjected to SDS-PAGE and immunoblotting using standard protocols (Gloy et al., 2002 (link)). Chemiluminescence was acquired by the ChemiDoc MP imager (BioRad) and band intensities were quantified by the accompanying software (BioRad).