Sedoheptulose bisphosphatase activity assay

HFFs were grown to deep quiescence in 60 mm dishes as before, then the media was changed to metabolic media for 35 hours. One hour before infection, the media was change to metabolomic media except using glucose free RPMI1640 supplemented with [6-¹³C₁]glucose at normal glucose concentration (2 g/L) (Sigma-Aldrich). Dishes in triplicate were infected with 2 X 10⁶ tachyzoites, or mock infected with an equal addition of media by volume. At 12 HPI for RH type I infections or 24 HPI for ME49 type II infections, dishes were quenched and metabolites extracted, processed, and analyzed as previously described in the “infection time course metabolomics” section, except that samples were resuspended in 75 μL of HPLC grade water and 20 μL was injected. MAVEN software was used to identify the isotopic forms of sedoheptulose-7-phosphate and sedoheptulose-1,7-bisphosphate, while correcting for the natural abundance of ¹³C.