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. 2015 Aug 3;112(33):E4581–E4590. doi: 10.1073/pnas.1510031112

Fig. 4.

Fig. 4.

Gbp2 positively regulates Irga6 recruitment to T. gondii through prenylation. (A) T. gondii numbers at 36 h postinfection in WT and Gbp2-deficient MEFs untreated or treated with IFN-γ were analyzed by luciferase assay. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated WT or Gbp2-deficient cells relative to those in the unstimulated respective cells are shown as “Relative T. gondii numbers.” Indicated values are means ± SD of triplicates. (B and C) The percentage of parasites positive for Irga6 (B) or Irgb6 (C) staining at the indicated points postinfection in IFN-γ–stimulated WT and Gbp2-deficient MEFs. Indicated values are means ± SD of triplicates. (D) Gbp2 mutants possessing amino acid substitutions in the GTPase domain or Caax motif. (E) Lysates of Gbp2-deficient MEFs stably transfected with empty or indicated HA-tagged Gbp2 expression plasmids were detected with indicated Abs by Western blot. (F and G) IFN-γ–stimulated Gbp2-deficient MEFs stably transfected with empty or indicated HA-tagged Gbp2 expression plasmids were infected with ME49 T. gondii (moi = 4), fixed at 1 h postinfection, and incubated with rabbit anti-T. gondii (green), mouse anti-HA (red), and DAPI (blue). (Scale bars, 10 μm.) Arrows indicate colocalization of Gbp2 with T. gondii. Percentage of parasites positive for Gbp2 staining is shown G. Indicated values are means ± SD of triplicates. (H) T. gondii numbers at 36 h postinfection in Gbp2-deficient MEFs stably transfected with empty (control) or indicated HA-tagged Gbp2 expression plasmids untreated or treated with IFN-γ were analyzed by luciferase assay. Percentages of parasite numbers (calculated by luciferase counts) in IFN-γ–stimulated control or indicated Gbp2 variants expressing cells relative to those in the unstimulated respective cells are shown as “Relative T. gondii numbers.” Indicated values are means ± SD of triplicates. (I) The percentage of parasites positive for Irga6 staining in Gbp2-deficient MEFs expressing indicated Gbp2 variants at 3 h postinfection. Indicated values are means ± SD of triplicates. N.D., not detected; N.S., not significant; **P < 0.01, ***P < 0.001. Data are representative of two (E and F) or three (A and H) independent experiments. Data in B, C, G, and I are pooled from two independent experiments, in which almost 150 cells at each time point were counted.