Ni2 sepharose beads

HB pull downs were performed as described elsewhere [5 (link)]. Cells were harvested by filtration, deep frozen and ground using a SPEX Freezer Mill 6870 (SPEX SamplePrep, Metuchen, NJ, USA) applying standard settings [5 (link)]. The cell powder was suspended in buffer 1 (6 M guanidine HCl, 50 mM Tris pH 8.0, 5 mM NaF, 1 mM PMSF, 2 mM sodium orthovanadate 0.1% Tween, protease inhibitor cocktail (Roche, Basel, Switzerland, 11,873,580,001), pH 8) and cleared by centrifugation (13,500×g, 15 min, 4 °C), incubated with Ni2 + −Sepharose beads (GE Healthcare, Buckinghamshire, UK, 17–5318-06) for 4 h at room temperature, washed with urea buffer (8 M urea, 50 mM sodium phosphate buffer pH 8.0 (and pH 6.3), 300 mM NaCl, 0.01% Tween 20). Proteins were eluted in urea buffer pH 4.3 containing 10 mM EDTA, incubated with streptavidin-agarose beads, washed with urea wash buffer containing 1% SDS and without SDS. Beads were re-buffered to 50 mM ammonium bicarbonate (ABC). Samples were reduced using DTT, Cys-residues were alkylated with 20 mM iodoacetamide (IAA), incubated with 300 ng trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) at 37 °C overnight, quenched with trifluoroacetic acid (0.5% final concentration) and desalted using C18 Stagetips [10 (link)].

The GBF1 coding sequence was cloned into pcDNA5/FRT/TO1/His-PC-TEV-Blue, to fuse His₆ and Protein C (PC) tags (followed by a TEV cleavage site) to the N-terminus of the GBF1 protein. HEK293 cells were transfected with this pcDNA5/FRT/TO1-His₆-PC-TEV-GBF1 construct to generate a stable HEK293 cell line by directed homologous recombination, as described previously (Derivery et al., 2009). His₆-PC-GBF1 expression was induced with 7 µg/ml of tetracycline for 40 h. Tandem affinity purification was carried out by preparing lysates from the His₆-PC-GBF1 HEK293 stable cell line in 200 mM NaCl, 1 mM CaCl₂, 1% Triton X-100, 5% glycerol, 50 mM Na–HEPES, pH 7.4, and incubating first with protein C beads (Roche) then Ni²⁺ Sepharose beads (GE Healthcare), as described previously^{47 (link)}. After extensive washing, Ni²⁺-Sepharose beads were boiled in SDS loading buffer, bands were cut from the gel, and then analyzed by mass spectrometry. Two subunits of the COPI coat were identified, validating the approach, as we have previously identified the COPI complex as an interacting partner of GBF1^{48 (link)}.

For HB pull‐downs, one liter of yeast cell culture was harvested by filtration, deep‐frozen, and ground using a SPEX Freezer Mill 6870 (SPEX SamplePrep) applying standard settings (Reiter et al, 2012 (link)). Lysed cells were resolved in buffer 1 (6 M guanidine HCl, 50 mM Tris pH 8.0, 5 mM NaF, 1 mM PMSF, 2 mM sodium orthovanadate 0.1% Tween, protease inhibitor cocktail (Roche), pH 8), cleared by centrifugation (13,500 × g, 15 min, 4 °C), incubated with Ni²⁺−Sepharose beads (GE Healthcare) for 4 h at room temperature, and washed with urea buffer (8 M urea, 50 mM sodium phosphate buffer pH 8.0 (and pH 6.3), 300 mM NaCl, 0.01% Tween 20). Proteins were eluted in urea buffer pH 4.3 containing 10 mM EDTA, incubated with streptavidin–agarose beads (Thermo Fisher Scientific), washed with urea wash buffer containing 1% SDS and without SDS. For on bead digests, samples were re‐buffered to 50 mM ammonium bicarbonate (ABC), reduced using DTT, treated with 20 mM iodoacetamide (IAA) to alkylate cysteine residues, incubated with 300 ng trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) at 37 °C overnight, quenched with trifluoroacetic acid (0.5% final concentration), and desalted using C18 StageTips (Rappsilber et al, 2007 (link)).