Peg maleimide

The PEG switch assay was performed according to a previously described protocol⁶¹ . In brief, cells were treated with 50 mM D-ala, 50 mM L-ala, 8 mM NAC or 500 µM H₂O₂ respectively. After a washing step with ice-cold PBS containing 20 mM of N-ethylmaleimide (NEM), 150 µL of ice-cold PEG switch lysis buffer containing 1% SDS, 100 mM NEM and 100 mM Tris pH 7.4 were added to the cells. Each lysate was incubated in a shaker at 50 °C and 600 rpm for 25 min to block free thiol groups by alkylation with NEM enriched lysis buffer. While 50 µL from each sample were used as inputs, the rest 100 µL were supplemented with buffer containing 200 mM DTT to reduce oxidized protein thiols. After a 30 min incubation at room temperature, excess DTT was removed using Zeba™ Spin desalting columns (Thermo-Fisher Scientific). After addition of the thiol-labeling buffer containing 70 mM PEG-maleimide (5 kDa; number 63187; Sigma-Aldrich) and 7% SDS in 500 mM Tris pH 7.4, samples were incubated on a laboratory agitator at room temperature for 2 hrs in the dark. Finally, each input and sample was supplemented with 6× reducing Laemmli sample buffer containing 5% ß-mercaptoethanol and subjected to Western immunoblot analysis.

Membrane fractions were resuspended and incubated in MELB supplemented with 0.5 mM PEG-maleimide (5 kDa, Sigma-Aldrich) and cOmplete protease inhibitors (Roche) at room temperature for 30 min. The reaction was then quenched by addition of n-ethylmaleimide at a final concentration of 20 mM. Proteins were solubilised in 1% w/v digitonin for 30 min at 4°C prior to immunoprecipitation of HA-VDAC2 with anti-HA beads (Thermo Fisher) for 1 h at 4°C. Proteins were eluted from the beads by boiling for 5 min in SDS-PAGE sample buffer prior to analysis on SDS-PAGE.