Fig. 4. TgMIC6 and TgMIC8 are microneme proteins, processed at their C-terminus upon secretion. Western blot analysis of T. gondii tachyzoites probed with antisera raised against NterMIC6 or the CterMIC6 (A) and with antisera raised against NterMIC8 and CterMIC8 (B). Lysates were prepared from freshly lysed parasites from RH, mic6ko in RH or Prugniaud (Pru) strains and from vero cells. (C) Western blot analysis of lysates from parasite pellets and supernatants of secretion assays (ESA). TgMIC6 is processed from a 53 kDa precursor into a 45 kDa product in the parasites during its transport to the micronemes. In the ESA, the 35 kDa form is detectable with -N-terMIC6 but failed to be recognized by the -CterMIC6 antibodies. (D) A similar analysis was performed on TgMIC8 and showed that the 65 kDa secreted form of TgMIC8 is not detectable with -CterMIC8, establishing that the C-terminal domain has been removed. (E) Immunofluorescent labeling of permeabilized HFF cells infected with tachyzoites from RH strain. Double labeling with mouse -MIC3 and rabbit -MIC8 confirmed the microneme localization of TgMIC8. The antibodies were visualized with Alexa^TM 488 goat -mouse IgG and Alexa^TM 594 goat -rabbit IgG antibodies. Bar, 1 µm.