Fig. 5. Complementation analysis of MIC8KOi using different chimeric proteins. (A) Schematic of the complementation strategy. Transfection of MIC8KOi with complementation constructs under ATc selection results in three different integration events: (1) homologous recombination leads to a promoter exchange resulting in constitutive expression of Ty-tagged MIC8 (stained in green); (2) a combination of both homologous and non-homologous recombination leads to constitutive expression of both MIC8Ty (stained in green) and the respective myc-tagged complementation construct (stained in red); (3) only non-homologous recombination leads to exclusive, constitutive expression of the myc-tagged complementation construct. This event indicates functional complementation. The immunofluorescence analysis shown was performed on a representative pool of MIC8KOi parasites complemented with pMIC8MIC8mycTMCTD^MIC8 after 6 days under ATc selection. (B) Top: overview of the complementation constructs used in this study. The TMD and CTD of MIC8myc have been substituted as indicated. In the case of MIC2, two constructs were generated (see text). In the case of MIC8mycGPI (GPI), the TMD and CTD of MIC8 was exchanged for the GPI anchoring signal of the major surface antigen SAG1. Bottom: representative immunofluorescence analyses of MIC8KOi transfected with the indicated complementation construct. Stable transfection has been achieved by application of ATc selection as shown in A. Numbers in each image indicate the average percentage of parasites (± s.d.) in which random integration occurred in four independent experiments. Scale bars: 12.5 µm.