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. 2011 Sep 29;7(9):e1002222. doi: 10.1371/journal.ppat.1002222

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Nebl 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) Immunofluorescence analyses of Toxoplasma parasite lines expressing HA-tagged ELC1 confirms the co-localization of this proteins with GAP45 at the parasite periphery, and (B) demonstrates a stable association with the inner membrane complex. (C-F) Immunoprecipitation analyses validate that ELC1 is an integral component of the intact MyoA invasion motor complex machinery. C) Western blot analyses of anti-HA immunoprecipitates prepared from detergent-soluble protein extracts of wild-type (WT) or transgenic parasites expressing HA-tagged MLC1 (MLC1-HA) or ELC1 (ELC1-HA) were probed by Western blot using antibodies against the HA epitope tag, as indicated. Arrows show the relative size of MLC1-HA (∼35 kDa) and ELC1-HA (∼18 kDa) protein bands. We also observed smaller immunoreactive MLC1-HA or ELC1-HA bands in HA pull downs (*). Western blot analyses using specific rabbit polyclonal antibodies against MyoA (top), GAP45 (center) or MLC1 (bottom) confirm the co-purification of other invasion motor complex components in anti-HA immunoprecipitates of parasite lines expressing MLC1-HA (lane 2) or ELC1 (lane 3), but not wild-type controls (lane 1). Arrows show the sizes of endogenous Toxoplasma MyoA, GAP45, MLC1, and the size of the immunoreactive band corresponding to MLC1-HA (lane 2, arrowhead). D) Immunoprecipitates of parasites expressing MLC1-HA were purified using magnetic microbeads coated with anti-HA antibodies and eluted proteins stained with Sypro Ruby. Major bands corresponding to MyoA, GAP50, GAP45, GAP40, and MLC1 were confirmed by LC-MS/MS. A ∼15 kDa protein band was precipitated in addition to the other known invasion motor complex components and were excised from a preparative 10% SDS-PAGE gel (arrow). This protein band yielded 44% sequence coverage for ELC1 (Supplementary Figure S8). E, F) Immunoprecipitates of parasites expressing ELC1-HA were prepared using anti-GAP45 antibodies. Eluted proteins were stained with Sypro Ruby (E) or probed by Western blot using anti-HA antibody (F). A ∼18 kDa protein band corresponding to ELC1-HA was detected in addition to the other known invasion motor complex components (E, arrow). Western blot analyses confirm the co-purification of ELC1-HA with motor complexes prepared from parasite lines expressing HA-tagged ELC1 using specific rabbit polyclonal antibodies against GAP45 (lane 2, arrow), but not in pull-downs prepared using non-specific rabbit IgG (lane 1). An asterisk indicates a putative degradation product, proteolytic fragment or posttranslational modification of ELC1-HA. G.) Structural model of Toxoplasma MyoA-tail (green) interacting with MLC1 (Cyan) and the newly identified ELC1 (Magenta) based on P. polycephalum myosin regulatory complex [34].