Protein extraction and western blotting

Protein extracts for western blot analysis, cell pellets (∼5 × 10⁶ cells) were resuspended in benzonase buffer (50 mM pH 7.5 Tris–HCl, 20 mM NaCl, 1 mM MgCl₂, 0.1% SDS, benzonase 0.01 U/ml) containing protease inhibitors, sonicated in a Bioruptor^® bath (pulses 30′ on/30′ off for 10 min at maximum intensity) and centrifuged at 12 000 rpm for 5 min at 4°C. When removal of soluble proteins was required, cell pellets (∼5 × 10⁶ cells) were incubated for 5 min at 4°C in 1 ml ice-cold CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES pH 6.8, 3 mM MgCl₂) containing 0.5% triton X-100 and protease inhibitors. After centrifugation at 12 000 rpm for 5 min at 4°C, pellets were washed twice with 1 ml ice-cold CSK. The resulting pellets were resuspended in Laemmli buffer and boiled. Between 20 and 40 μg of proteins were loaded onto 4–15% Mini-PROTEAN TGX Precast Protein Gels (Biorad). Proteins were separated by electrophoresis at 30 mA and transferred to Trans-Blot membranes (Biorad) for 10 min at 1.3A using a Trans-Blot® Turbo machine (Biorad). Before addition of primary antibodies, membranes were blocked overnight in PBS containing 0.1% Tween 20 (PBST) and 3% milk. The next day, the membrane was washed three times with 1× PBST and incubated with the primary antibody for 1 h at room temperature. Antibodies used are indicated in Supplementary Table S4. After three washes with 1× PBST, the membrane was incubated with the secondary antibodies for 1 h at room temperature. Western blots were revealed with the ODYSSEY CLx.