Dimethyl pimelimidate

Antibodies against Slc1 and Mcsc were crosslinked separately to protein A/G resins (Pierce, Rockford, Illinois, USA) using 40 mM Dimethyl pimelimidate (Sigma-Aldrich, St. Louis, Missouri, USA) and the reactions were quenched with 40 mM ethanolamine (Sigma-Aldrich, St. Louis, Missouri, USA). Pre-immune serum was also crosslinked to resins and served as a negative control. Approximately 3×10¹⁰ EBs were lyzed by sonication in 1 mL Pierce IP lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol; pH 7.4)(Pierce, Rockford, Illinois, USA) supplemented with 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1X EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland). After centrifugation to pellet down insoluble debris, the lysate was incubated with the antibody-crosslinked resins at 4°C for 4 hours. After 3 washes with IP lysis buffer, the bound proteins were eluted with Pierce Elution buffer (pH 2.8) (Pierce, Rockford, Illinois, USA) and separated via SDS-PAGE on a 4–12% Bis/Tris gradient gel (Invitrogen Life Technologies, Carlsbad, California, USA). Lanes were sliced into 8–10 equivalent sections for in-gel digestion and analyzed by mass spectrometry at the Duke Proteomics Core facility.

IPs were performed as previously described31 (link). Briefly, 4 μg of Mitofilin, CHCHD6 or negative control antibody was crosslinked to protein A Sepharose beads using dimethylpimelimidate (Sigma) at a 1:1 ratio of beads to antibody. HeLa cells were lysed in IP lysis buffer (Beyotime, China) containing protease inhibitor EDTA-free (Roche) for 30 min at 4 °C followed by centrifugation at 14 000 g for 30 min at 4 °C. The supernatant was then incubated overnight with 50 μl antibody-crosslinked beads at 4 °C. The immunocomplex was collected, washed five or six times with cold PBS, and mixed with protein gel loading buffer to elute the proteins at room temperature. Mitofilin and CHCHD6 immunoprecipitates were trichloroacetic acid (TCA) precipitated and sent to BGI TechSolutions Co., Ltd., Shenzhen, China, for LC-MS/MS analysis.

Purified human ceruloplasmin (Alexis Biochemicals) was added to CSFs (20 μg/ml, final concentration) and incubated at 37 °C to the indicate time points under nitrogen-conditioned atmosphere, to avoid the exposure to atmospheric oxidative environment; in selected experiments, incubation was performed in presence of catalase (60 μg/ml, Sigma-Aldrich). After incubation in CSFs, spiked ceruloplasmin was immunoprecipitated using protein-G agarose beads (Invitrogen) coated with polyclonal sheep anti-ceruloplasmin antibody (Ab) (ab8813 Abcam, raised against full length purified ceruloplasmin) cross-linked with 20 mM dimethyl-pimelimidate (Sigma-Aldrich). This polyclonal Ab anti-ceruloplasmin was selected for its ability to equally immunoprecipitate both resting- and oxidized-ceruloplasmin (see Additional File 1: Figure S2). Ceruloplasmin was eluted with 0.1 M glycine, pH 2.5, and diafiltered (Amicon-Millipore) in PBS. In vitro oxidation and deamidation of ceruloplasmin (Cp-ox/AmBic) was achieved by incubation (16 h at 37 °C) in 100 mM ammonium bicarbonate buffer pH 8.5, containing 10 mM hydrogen peroxide [14 (link)].

Mouse control IgG (SCBT, sc-2025) were covalently coupled to Sepharose-protein A/G (SCBT, sc-2003) beads using dimethylpimelimidate (Sigma-Aldrich, D8388). Briefly, 200 μL of Sepharose-protein A/G was washed with 1× PBS twice, incubated with 200 μL of antibody (20 μg) solution (1× PBS) for 1 hour at room temperature. Antibody-bound protein A/G beads were then incubated with 1% chicken egg ovalbumin in PBS for another hour to block nonspecific binding sites. After three washes with 1× PBS, 25 mg of dimethylpimelimidate in 1 mL of 200 mM triethanyl amine was added, and coupling reaction proceeded at room temperature for 30 minutes. The reaction was repeated 2 more times with fresh addition of dimethylpimelimidate and quenched with 50 mM ethanolamine. The reacted protein G beads were washed extensively with 1× PBS before immunoprecipitation.

The cells were lysed using a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 M EDTA, 10 mM sodium glycerophosphate, 10 mM sodium pyrophosphate, 10% glycerol, 1% NP-40, 0.5 mM PMSF, 5 mM NaF, 1 mM Na₂MoO₄, 0.5 mM NaVO₄, and protease inhibitor cocktail (Roche). The cell lysates (1–2 mg) were precleared by incubation with 10 μL of protein G–sepharose (GE Healthcare Dharmacon, Chicago, IL, USA) at 4 °C for 30 min. The lysate extracts were then incubated overnight at 4 °C with 5–10 μL of protein G–sepharose that was covalently coupled to 1.2 μg of the primary anti-TRIB3 (Abcam, ref. #75946, Cambridge, UK) or an unspecific IgG (negative control) using dimethyl pimelimidate (Sigma-Aldrich, St. Louis, MO, USA). The immunoprecipitates were washed 3 times with HEPES lysis buffer followed by 1 wash with HEPES kinase buffer (25 mM HEPES pH 7.5 and 50 mM KCl) and resuspended in 20 μL of sample buffer. The samples were subjected to electrophoresis and immunoblot analysis following standard procedures.

His-tagged Arl13b-C-ter was purified under denaturing conditions using 8 M urea, as previously described (Mahajan et al., 2013 (link)). The denatured protein was used to immunize rabbits, and anti-sera were collected by Genemed Synthesis Inc. To purify the Arl13b antibody from the anti-serum, GST–Arl13b-C-ter on glutathione Sepharose beads were prepared as previously described (Mahajan et al., 2013 (link); Zhou et al., 2013 (link)) and incubated with dimethyl pimelimidate (Sigma) in 200 mM sodium borate solution pH 9.0 to cross-link the fusion protein to glutathione. After blocking the excess cross-linker with ethanolamine, the cross-linked beads were incubated with anti-serum at room temperature. The beads were subsequently washed with PBS, and the bound antibody was eluted by using 100 mM glycine pH 2.8. The pH of the eluate was adjusted to neutral immediately, and the eluted antibody was dialyzed, concentrated, quantified and stored at −80°C.

Magnetic Dynabeads (Life Technologies, 10004) were incubated with Rabbit anti-FLAG (NEB, 14793S) in PBS-0.01% Tween 20 (Thermo Fisher Scientific, 10485733) rotating for 15 min at room temperature. Dynabeads were then cross-linked with dimethyl pimelimidate (Sigma-Aldrich, D8388) in 0.2 M triethanolamine (Sigma-Aldrich, 90279) pH 8.2 rotating for 30 min at room temperature. The cross-linking reaction was subsequently stopped with 50 mM Tris-HCl (Sigma-Aldrich, T5981) pH 7.5 rotating for 15 min at room temperature.