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Synchronized ring stage parasites were exposed to either agent at 2 hours post synchronization for 8 hours with RNA extraction at 10 hours post synchronization.
Parasite morphology was evaluated by examination of blood smears every 6 hrs until reinvasion, i.e. 52 hrs post synchronization.
The timing of this analysis at 10 hours post synchronization coincides with the peak of var gene expression determined by a time-curve assay (data not shown).
The peak of GSK3β phosphorylation correlates with the increase in BMAL1 protein levels; vice versa, when GSK3β activity is high (24 to 30 hours post synchronization), BMAL1 levels are low (Fig. 4A,C).
Using anti phospho-GSK3β antibodies we reveal that there is more phosphorylation of GSK3β (corresponding to decreased kinase activity) at 6 to 12 hours post synchronization of the MEFs (Fig. 4C).
Ring-stage parasites (6 hours post synchronization) were exposed to two forms of stress inducing conditions: oxidative stress (10 µM of tert-butylhydroperoxide [tBHP] for 4 hours) and glucose deprivation (4 hour culture in the presence of 1.95 g/l glucose, 50% of the normal concentration), immediately followed by RNA extraction.
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With the parents in the film, the post-synchronization is not so good.
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