Gsh agarose beads

S. cerevisiae Arx1 was expressed as an N-terminal GST fusion from pGEX-6P-1 in Escherichia coli Rosetta (DE3) pLysS cells. Five hundred milliliters of LB medium (+100 µg/ml ampicillin and 40 µg/ml chloramphenicol) were inoculated to a starting OD₆₀₀ 0.04 and grown at 37 °C (170 r.p.m.). At an OD₆₀₀ of 0.4, the culture was shifted to a shaking water bath at 16 °C, and after 30 minutes, heterologous protein expression was induced with 0.4 mM IPTG for 18 hours. Collected cells were washed once with aqua bidest and stored at −80 °C. For cell lysis, cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 1× HP protease inhibitor cocktail (Serva)) and incubated with 1 mg/ml lysozyme for 45 minutes prior to sonification. After removal of cell debris by centrifugation (40,000g, 25 min, 4 °C), the supernatant was incubated with GSH-agarose beads (Sigma Aldrich) at 4 °C for 60 minutes. Afterwards, beads were washed twice with lysis buffer, once with washing buffer (50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM DTT) and twice with storage buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% vol/vol glycerol, 1 mM DTT). The beads with the bound GST-Arx1 were resuspended in storage buffer, portioned, shock frozen in liquid nitrogen and stored at −80 °C.

GST-tagged wild-type Drg1 and the EQ1 (E346Q) variant were overexpressed in yeast as described^{12 (link),16 (link)}: the expression strain was inoculated to a starting OD₆₀₀ of 0.01, incubated at 30 °C at 110 r.p.m. in baffled flasks. Protein expression was induced by immediate addition of 0.025 µM CuSO₄, and cells were collected after 24 hours. Affinity purification was performed as described^{12 (link),16 (link)–18 (link)}: frozen cells were thawed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1 mM DTT, 1× complete protease inhibitor cocktail (Roche) and 0.5 mM PMSF) and disrupted by vigorous shaking with 0.6 mm glass beads in a beadmill (Merckenschlager). Homogenates were centrifuged twice at 40,000g at 4 °C for 15 and 30 minutes, respectively. Crude extracts were incubated for 90 minutes at 4 °C with GSH-agarose beads (Sigma Aldrich) for affinity purification of GST-tagged Drg1. After consecutive washing steps (3× with lysis buffer plus 1 mM EDTA, 1 mM DTT and 1× with elution buffer plus 1 mM DTT), the protein was eluted. For elution, the GST tag was cleaved off using Prescission protease (GE Healthcare/Cytiva) overnight at 4 °C on a rotator in elution buffer suitable for the respective experiment. Protein concentration was measured using the Bradford assay (Biorad) with BSA calibration curve.

TUBE pull-down assay was performed as previously described.^{35 (link)} Cells were lysed in buffer (50 mM NaCl, 20 mM Tris pH 7,5, 1% NP40, 5 mM EDTA, 10% glycerol) supplemented with a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) for 20 min on ice. In all, 1000 μg of protein were incubated overnight at 4 °C with 50 μl GSH-agarose beads (Sigma-Aldrich) linked to GST-TUBE. Afterward beads were washed with buffer. The precipitate was analyzed for ubiquitin expression by Western blotting using mouse IgG1 anti-ubiquitin (P4D1) (Santa Cruz Biotechnology) antibody and for interaction with NOXA and MCL-1 by Western blotting using rabbit anti-MCL-1 antibody (ENZO), and mouse anti-NOXA antibody (ENZO). To verify the protein amount per lane, the membrane was stained with Ponceau S (AppliChem GmbH, Darmstadt, Germany).