2.6.2. Preparation of Cell Extracts

Extracts were prepared from HeLa cells incubated with 0, 50, 100 or 200 μM vitamin C for 6 h (3 h + wash + 3 h). They were washed with cold PBS, trypsinized, centrifuged at 800× *g* for 5 min at 4 °C and resuspended in cold PBS at a concentration of 1.25 × 10<sup>6</sup> cells per mL. Then the cell suspension was split into aliquots of 1 mL and centrifuged at 14,000× *g* for 5 min at 4 °C. The supernatant of each aliquot was completely discarded and the dry pellets were frozen in liquid

nitrogen and stored at −80 °C. After thawing the pellets were resuspended in 32 μL of extraction buffer A (45 mM HEPES, 0.4 M KCl, 1 mM EDTA, 0.1 mM dithiothreitol and 10% glycerol, pH 7.8) containing 0.25% of Triton X-100, mixed using vortex at high speed for 5–10 s, incubated for 5 min on ice and centrifuged at 14,000× *g* for 5 min at 4 °C. Then 27 μL of the supernatant was mixed with 110 μL of cold reaction buffer F.
