Figure 3. Allelic diversity and kinase activity of ROP17.

(A) Phylogenic analysis and neighbor joining tree of ROP17 sequences revealed two alleles in the three clonal lineages (i.e. Types I, II, and III). Scale = substitutions per site. (B) Cumulative nonsynonymous (nonsyn) and synonymous (syn) polymorphisms in ROP17 genes from A. (C) Proportion of nonsynonymous (pNS) vs. synonymous (pS) substitutions for the ROP17 alleles. (D) Kinase activities of the purified TAP complexes vs. rROP17. Casein (20 μg) (arrow) was incubated in a kinase reaction with approximately 2 ng of ROP17-TAP and ROP5-TAP, or alternatively 1 μg of rROP17, for 30 min in the presence of ³²P-γ-ATP. Kinase reaction products were resolved on 8-16% SDS-PAGE gels followed by transfer onto nitrocellulose and phosphorimager analysis. (E-F) ROP17 immunoprecipitated (IP) from wild type (Δku80) or mutant parasite lysates was incubated in a kinase reaction with the heterologous substrate dMBP (E) or Irgb6 (F) in the presence of radiolabelled ³²P-γ-ATP. Reaction products were resolved by 8-16% SDS-PAGE followed by transfer to nitrocellulose and phosphorimager analysis. Total parasite lysates were separated on 8-16% SDS-PAGE gels, Western blotted (WB) with mouse anti-MIC2 (Mo α-MIC2) and rabbit anti-ROP5 (Rb α-ROP5), and imaged on the LI-COR Odyssey Imaging System. The * in F likely denotes heavy chain IgG . Representative of three similar experiments.