The effect of PfCK2 knockdown on PfRh4 phosphorylation.

<p>(A) PfCK2-HA-DD retains kinase activity. PfCK2-HA-DD was immuno-precipitated from parasite lysate using an anti-HA antibody. <i>In vitro</i> kinase assay was performed using the immune-precipitatied eluate with the absence or presence of heparin. (B) Time course PfCK2 expression in 3D7-PfCK2DD parasites in the presence and absence of Shield-1. Shield-1 was removed at the ring stage of a synchronous parasite population, Day 0 (5% parasitemia). Absence of Shield-1 resulted in the progressive down-regulation of PfCK2. Parasite proteins were extracted using saponin lysis and analysed by western blotting for the presence of HA-tagged PfCK2 protein. HSP70 served as a loading control for normalisation. Densitometry analysis was performed and shown in bottom graph. (C) Growth assay of PfCK2-HA-DD in the presence and absence of Shield-1 (n = 2, error bars are standard error of the mean (SEM). Identical experiments were performed on a second cloned parasite line and similar results were obtained (n = 2). Parasitemia was quantified by counting Giemsa-stained smears. (D) Progression of PfCK2-HA-DD from late schizont to ring formation with and without Shield-1. Distinct ring, trophozoite and schizont populations were monitored using Sybr Green staining as the parasite progressed from 44 to 50 hours. One representative experiment is shown and was repeated 3 independent times. (E) Progression of PfCK2-HA-DD from late schizont to ring formation with and without Shield-1. Distinct schizont, late segmenter and ring populations were monitored using light microscopy and Giemsa-stained slides as the parasite progressed hourly from 42 to 48 hours. This experiment was performed independently twice and error bars shown are standard error of the mean (SEM).</p>