Irgm3 deficiency reverts the IRG recruitment and clearance defects of both Δ<i>rop18</i> and Δ<i>rop5</i> parasites <i>in vitro</i>.

<p>(A) Western blot analysis of wild type (RH<i>Δku80</i>), ROP18 deficient (RH<i>Δku80Δrop18</i>) and ROP18 complemented (RH<i>Δku80Δrop18</i>Complement) parasites. Western blot probed with rabbit anti-ROP18 and rabbit anti-actin flowed by goat anti-rabbit conjugated to HRP and detected by ECL. (B) Survival of female outbred CD1 mice challenged with 100 parasites by i.p. inoculation. n = 5 animals per group from a single experiment. <i>In vitro</i> clearance of parasites in wild type C57BL/6 (WT) (C) or Irgm3 deficient (Irgm3^−/−) (D) bone marrow derived macrophages following IFN-γ activation (50 units/mL). Means ± S.D. n = 6 samples from 2 combined experiments. For statistical analysis, parasite survival in Irgm3 deficient cells was compared to survival in wild-type cells. Student's <i>t</i> test, **<i>P</i><0.001. Quantification (E) and immunofluorescence localization (F) of Irgb6 localization to the vacuolar membrane in WT or Irgm3^−/− IFN-γ-activated macrophages. (E) Means ± S.D. n = 6 samples from 2 combined experiments. Student's <i>t</i> test, *<i>P</i><0.005. (F) The vacuole membrane was visualized by staining with mAb Tg 17-113 for GRA5 (secondary: Alexa Fluor 594, red). Irgb6 was visualized with rabbit-Irgb6 (secondary: Alexa Fluor 488, green). Scale = 5 microns.