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. 2012 Dec 14;7(12):e51773. doi: 10.1371/journal.pone.0051773

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© 2012 Coppens et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Panel a: Immunoblots of homogenates from wild-type T. gondii (left lane) and HFF (right lane) revealed with anti-CAV1 antibodies (α-CAV1), showing no expression of CAV1 by the parasites as confirmed by IFA in Panel b. (B) Expression of HA-CAV1 by transgenic parasites. Immunoblots of homogenates from wild-type T. gondii (left lane) and Toxoplasma transiently expressing HA-CAV1 for 36 h (right lane) revealed with anti-HA (α-HA), showing a band at ∼24-kDa in transgenic parasites. Panel b: Localization of HA-CAV1 in transgenic parasites by IFA showing a fluorescent punctuate pattern in transgenic parasites. (C) Panel a: Expression of CAV1-YFP by transgenic parasites. Immunoblots of homogenates from HFF infected with parasites stably expressing CAV1-YFP (left lanes) and uninfected HFF (right lanes) revealed with antibodies against CAV1 or against GFP (α-GFP) as indicated. The Western blots reveal bands at 48-kDa and 24-kDa corresponding to CAV1-YFP in T. gondii and CAV1 in HFF, respectively. Panel b: Localization of CAV1-YFP in transgenic parasites. Left: Direct fluorescence microscopy assays of CAV1-YFP-expressing parasites showing a vesicular staining. Right: Double immunofluorescence assays (IFA) of CAV1-YFP-expressing parasites using anti-CAV1 and anti-GFP antibodies illustrating structures co-labeled with the two antibodies. (D) Post-translational palmityolation of CAV1 in transgenic parasites. Panel b: Infected HFF with HA-CAV1-expressing parasites were labeled with [¹⁴C]palmitate for 6 h before isolation of the parasites. Toxoplasma homogenates were then immunoprecipitated with anti-HA antibodies and resolved in SDS gels containing either Tris or Tris/hydroxylamine for 12 h before analysis for fluorography. The gels shown are representative of one on two separate experiments, and revealed the association of radioactive palmitate with HA-CAV1. Panel b: Localization of mutant CAV1-YFP lacking palmitoylation residues in transgenic parasites. Direct fluorescence assays of intracellular Toxoplasma expressing CAV1^C133A-YFP (CAV1^C133A), CAV1^C143A-YFP (CAV1^C143A) or CAV1^C156A-YFP (CAV1^C156A) showing more cytosolic staining for YFP compared to Toxoplasma expressing wild-type CAV1.