<p>(A) <i>In vitro</i> kinase assays of wildtype and putative phosphosite mutations in PfRh4 cytoplasmic domains. The amino acid sequence of the PfRh4 cytoplasmic tail is shown with serines and tyrosines highlighted with the residue number. Each potential phosphosite on the PfRh4 tail was individually mutated to alanine. The all 4 lane has mutations in S1667A, S1674A, Y1680A and Y1684A and the all 5 lane has S1652A, S1667A, S1674A, Y1680A and Y1684A putative kinase sites mutated. The phosphorylation signal was quantitated and adjusted for protein loading. The loading-adjusted mutant phosphorylation signals were divided by the wildtype and plotted as a percentage of the wildtype signal (Y-axis). Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie gel from which protein loading was quantitated are shown in lower panels. Lane labels (X-axis) denote residues mutated to alanine. Mean percentage of wildtype phosphorylation +1 standard error of the mean are displayed. Data was averaged from four experiments performed on separate days. (B) Dosage-response curve for PfRh4 tail phosphorylation by merozoite lysate in the presence of increasing concentrations of the CK2 inhibitor TBB. PfRh4 tail phosphorylation was quantitated after incubation in <i>in vitro</i> phosphorylation assay with TBB. The phosphorylation signal for each condition was adjusted to reflect the average amount of protein loaded across each condition, determined by densitometry of the Coomassie brilliant blue stained gel. Y-axis represents loading-adjusted phosphorylation signal as a percentage of phosphorylation in the presence of DMSO (control). Autoradiograph of wildtype GST-fused PfRh4 proteins after incubation in the <i>in vitro</i> phosphorylation assay. X-axis indicates the TBB concentration with which the phosphorylation assay was incubated or DMSO. (C) <i>In vitro</i> kinase assays of PfRh and EBL cytoplasmic tails. The phosphorylation signal was quantitated and adjusted for protein loading. Autoradiograph of proteins after incubation in the <i>in vitro</i> phosphorylation assay and Coomassie brilliant blue stained gel from which protein loading was quantitated are shown. Data was averaged from four experiments performed on separate days (right panel) and standard error of the mean is shown. The following sites were mutated: EBA140 (S1159A, S1168A, T1173A), EBA175 (T1466A, mut A) and (S1489A, mut B) and in combination (mut A and B), EBA181 (S1528A, S1553A, S1557A, T1564A), PfRh2a (S3128A) and PfRh2b (S3233).</p
