TY - DATA T1 - The effect of PfCK2 knockdown on PfRh4 phosphorylation. PY - 2016/01/05 AU - Wai-Hong Tham AU - Nicholas T. Y. Lim AU - Greta E. Weiss AU - Sash Lopaticki AU - Brendan R. E. Ansell AU - Megan Bird AU - Isabelle Lucet AU - Dominique Dorin-Semblat AU - Christian Doerig AU - Paul R. Gilson AU - Brendan S. Crabb AU - Alan F. Cowman UR - https://plos.figshare.com/articles/figure/_The_effect_of_PfCK2_knockdown_on_PfRh4_phosphorylation_/1629146 DO - 10.1371/journal.ppat.1005343.g003 L4 - https://ndownloader.figshare.com/files/2615087 KW - PfRh 4 cytoplasmic domain KW - Plasmodium falciparum Adhesins Play KW - malaria KW - type 1 membrane proteins KW - PfRh 4 cytoplasmic tail KW - erythrocyte KW - cytoplasmic tails KW - merozoite KW - Live Cell Imaging KW - invasion KW - ebl KW - parasite Plasmodium falciparum N2 - (A) PfCK2-HA-DD retains kinase activity. PfCK2-HA-DD was immuno-precipitated from parasite lysate using an anti-HA antibody. In vitro 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). ER -