Cellular nucleotide extraction and Ap4A determination were performed using a sensitive luminescence-based assay. Briefly, nucleotides were extracted from cells by adding 5 ml of ice-cold trichloroacetic acid and subsequent neutralization with an equal volume of 0.6 M tri-octylamine in 1,1,2-trichlorotrifluoroethane. Following centrifugation at 500g for 5 min, the top aqueous layer was removed, and 10 U of thermo-sensitive alkaline phosphatase (Thermo Fisher Scientific) was added to degrade adenosine triphosphate (ATP). Remaining nucleotides were concentrated using DEAE-Sephacel beads. Following mixing and centrifugation at 12,000g for 1 min, beads were washed with water. Nucleotides bound to DEAE-Sephacel were eluted twice with 1 M triethylammonium bicarbonate (pH 8.5), and eluates were vacuum-dried. Nucleotides from the two eluates were combined and dissolved in 120 μl of Ap4A assay buffer [25 mM Hepes-NaOH (pH 7.8) and 5 mM magnesium acetate]. To remove any remaining ATP, a further 10 U of alkaline phosphatase was added, incubated at 37°C, and subsequently incubated at 95°C to denature alkaline phosphatase. Ap4A levels in 10 μl of sample were determined by adding 50 μl of Bactiter GLO (Promega). Background ATP levels were first measured on a Berthold Lumat 9507, and once stabilized, recombinant Ap4A hydrolase was added to cleave Ap4A (ATP + AMP), and levels of ATP were measured by the increase in luminescence. Levels of Ap4A in samples were determined by comparison to standards and expressed as pmol per 106 cells.

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