Cell lysis

Cells were harvested by washing twice with phosphate-buffered saline (PBS) and scraping into ice-cold lysis buffer containing 50 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, 1.15 mM sodium molybdate, 4 mM sodium tartrate, 10 mM β-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate and 1x complete protease inhibitor cocktail (Roche). After incubation for 10 min on ice, lysates were cleared by centrifugation at 20,000 x g for 10 min at 4°C. Protein concentration was determined using Bradford protein assay. For chemical cross-linking by DSP, cells were treated with compounds for 2 h before lysis. For this, after washing cells with pre-warmed PBS twice, cells were incubated with pre-warmed PBS containing 1 mM DSP and compounds for further 30 min at 37°C. The reaction was quenched by adding 20 mM Tris-HCl in PBS for 20 min. Cells were then lysed in lysis buffer without reducing agents.