ROP5 regulates the kinase activity of ROP18.

<p>(A) Expression of ROP18 and ROP5 detected by western blotting of parasite lysates with rabbit anti-ROP18 (Rb α-ROP18), rabbit anti-ROP5 (Rb αROP5), and rabbit anti-actin (Rb αACT1) as a loading control. Representative of 3 experiments with similar outcomes. (B) Quantification of ROP18 expression by phosphorimager analysis of western blots, normalized for loading by actin staining. Means ± S.E.M. n = 3 experiments. (C) Immunofluorescence localization of ROP18 on the parasitophorous vacuole membrane in wild type (RH<i>Δku80</i>) and ROP5 deficient (RH<i>ΔKu80Δrop5</i>) parasites infecting HFF cells <i>in vitro</i>. ROP18 was localized based on the C-terminal Ty-1 epitope described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002992#ppat.1002992-Taylor1" target="_blank">[7]</a> using mAb BB2 (directly conjugated to Alexa Fluor 488, green). The vacuole membrane was stained with polyclonal rabbit anti-GRA7, described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002992#ppat.1002992-Dunn1" target="_blank">[64]</a>, (secondary: Alexa Fluor 594, red). Scale = 5 microns. (D) Immunofluorescence localization of ROP5 on the parasitophorous vacuole membrane in wild type (RH<i>Δku80</i>) and ROP5 deficient (RH<i>Δku80Δrop5</i>) parasites infecting HFF cells <i>in vitro</i>. The vacuole membrane was labeled with mAb Tg 17-113 for GRA5 (secondary: Alexa Fluor 594, red). ROP5 was labeled with polyclonal Ab for ROP5 (secondary: Alexa Fluor 488, green). Scale = 5 microns. (E) <i>In vitro</i> kinase reaction using the kinase domain of ROP18 (ROP18-KD, 100 ng) and the heterologous substrate dMBP ± recombinant ROP5 (rROP5, 200 ng). (F) <i>In vitro</i> kinase reaction using full length ROP18 (ROP18-FL, 25 ng) and the natural substrate Irgb6 ± recombinant ROP5 (rROP5, 50 ng). Irgb6 was immunoprecipitated (∼10–20 ng/reaction) from IFN-γ activated RAW cells with polyclonal rabbit anti-Irgb6. (G) Immunoprecipitation of ROP18 (∼5 ng/reaction) from parasite lysates with polyclonal rabbit anti-ROP18 (Rb anti-ROP18). Bound (denoted as B) and unbound (denoted as UB) samples were resolved by SDS-PAGE and blotted for ROP18 (Rb anti-ROP18 biotin). (H) <i>In vitro</i> kinase reaction of ROP18 using the heterologous substrate dMBP. ROP18 immunoprecipitations from (G) were incubated with or without substrate in the presence of ³²P ATP. Reactions were resolved by SDS-PAGE and subjected to phosphorimager analysis. (I) Immunoprecipitation of ROP18 (∼5 ng/reaction) from parasite lysates with rabbit anti-ROP18 as in (G). (J) <i>In vitro</i> kinase reaction of ROP18 and a natural substrate Irgb6. ROP18 immunoprecipitations from (I) were incubated with or without substrate in the presence of ³²P ATP. Irgb6 was immunoprecipitated from IFN-γ activated RAW cells with polyclonal rabbit anti-Irgb6. Reactions in E, F, H, and J were carried out in the presence of ³²P ATP, resolved by SDS-PAGE and subjected to phosphorimager analysis. E–J are representative of 3 or more experiments with similar outcomes.