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. 2015 Jul 31;11(7):e1005079. doi: 10.1371/journal.ppat.1005079

Fig 1. Detection and localization of EhVps32 protein in trophozoites lysates and fixed trophozoites.

Fig 1

(A) At left, Ponceau stained nitrocellulose membrane of transferred 12% SDS-PAGE separated trophozoites lysates (lanes 1 and 2) and molecular markers (lane M). At right, western blot assay using αrEhVps32 antibodies (lane 1) or preimmune serum (lane 2), followed by HRP-labeled secondary antibodies. (B) Western blot assays of trophozoites extracts (T), membrane fraction (M) and cytoplasmic fraction (C). Membrane fraction was ultracentrifuged to separate plasma membrane (PM) and internal membranes (IM). (C) Confocal microscopy using αrEhVps32 and αGal/GalNac lectin antibodies, followed by FITC-labeled and Pacific blue-labeled secondary antibodies, respectively. Controls: non-permeabilized trophozoites (NP) and trophozoites treated with preimmune serum (PS) and secondary antibodies. Nuclei were counterstained with DAPI (blue fluorescence). Arrows: plasma membrane. Arrowheads: vesicles close to plasma membrane magnified in white square. (D) TEM of trophozoites incubated with αrEhVps32 antibodies or PS and gold-labeled secondary antibodies. V: vesicles. Arrows: gold particles. Arrowheads: vesicle membrane. PM: plasma membrane.