Cell Lysis, Digestion, and Protein Fractionation

Cell lysis and digestion was carried out as described [15] . Briefly, cells were collected by centrifugation at 300g for 5 min, washed twice with ice cold PBS supplemented with phosphatase inhibitors (1 mM Na₃VO₄ and 1 mM NaF) and lysed with a denaturing buffer (20 mM HEPES pH 8.0, 8 M urea, 1 mM Na₃VO₄, 1 mM NaF, 2.5 mM Na₄P₂O₇, 1 mM ß-glycerol-phosphate) at a concentration of 10 × 10⁶ cells/mL. Cell lysates were further homogenized by sonication and insoluble material was removed by centrifugation at 20,000g for 10 min. Protein concentration in the supernatants was calculated by Bradford analysis and for each sample 0.5 mg of protein were resuspended in a volume of 1 mL of denaturing buffer. For linearity and accuracy assessment, control and treated cell lysates were mixed to a final protein concentration of 0.5 mg/mL. The proportions used were 0%, 25%, 50%, 75% and 100% of (pV) treated extracts mixed with 100%, 75%, 50%, 25% and 0% of vehicle treated extracts, respectively. For reduction and alkylation, protein mixtures were sequentially incubated with 4.1 mM DTT and 8.3 mM iodoacetamide for 15 min. For digestion, samples were diluted to 2 M urea with 20 nM HEPES pH 8.0 and incubated with immobilized TLCK-trypsin (20 TAME units/mg) for 16 h at 37 °C. Digestion was stopped by addition of TFA at a final concentration of 1%.