15 OD units of cells grown in exponential phase were washed with ice-cold PBS and kept on ice throughout the experiment. Cells were resuspended in 500 µL cold PBS and 300 µL was added to 2 × 2 mL bead beater tubes. 12.5 µL BMOE (125 mM in DMSO to a final concentration of 5 mM) or 12.5 µL DMSO was added before incubating on ice for 6 min. Cells were washed twice with 1 mL cold PBS containing 5 mM DTT.
Crosslinked cells were resuspended in 500 µL lysis buffer (150 mM NaCl; 5 mM EDTA; 0.5% [v/v] NP40; 500 mM Tris-HCl, pH 7.5; 1 mM PMSF; 1 tablet/50 mL protease inhibitor cocktail [Roche]; 1 mM DTT) and an equal volume of acid-washed glass beads (425–600 μm, Sigma) was added. The cells were lysed using a FastPrep−24 benchtop homogeniser (M.P. Biomedicals) at 4°C for 3 × 60 s at 6.5 m/s with a 5 min rest between cycles, until >90% of the cells were lysed as confirmed microscopically. The insoluble fraction was pelleted by centrifugation at 13,200 rpm for 10 min and the supernatant isolated and analysed by western blot.
Crosslinked cells were resuspended in 500 µL lysis buffer (150 mM NaCl; 5 mM EDTA; 0.5% [v/v] NP40; 500 mM Tris-HCl, pH 7.5; 1 mM PMSF; 1 tablet/50 mL protease inhibitor cocktail [Roche]; 1 mM DTT) and an equal volume of acid-washed glass beads (425–600 μm, Sigma) was added. The cells were lysed using a FastPrep−24 benchtop homogeniser (M.P. Biomedicals) at 4°C for 3 × 60 s at 6.5 m/s with a 5 min rest between cycles, until >90% of the cells were lysed as confirmed microscopically. The insoluble fraction was pelleted by centrifugation at 13,200 rpm for 10 min and the supernatant isolated and analysed by western blot.