Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2011 Sep 1;7(9):e1002197. doi: 10.1371/journal.ppat.1002197

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Vera 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.

PMC Copyright notice

(A) Immunofluorescence assay was performed on methanol fixed and permeabilized parasites post hepatocyte invasion at 2 hrs and 6 hrs development. The parasitophorous vacuole membrane (PVM) was detected by staining with α-pyUIS4. Parasites (both extracellular and intracellular) were stained with pyCSP. The percent UIS4-CSP double stained parasites was determined by counting parasites staining with both UIS4 (PVM staining; see B) and CSP and dividing this number by the number of parasites staining with CSP (total parasites including extracellular parasites). Numbers are the average of triplicates ± SEM (at least 300 parasites counted) and were statistically significant using the unpaired t test analysis. (B) Images from experiment in (A) of sporozoites at 2 hours post Hepa1–6 infection showing localization of PyUIS4 (red) within PVM and the associated tubulovesicular network (asterisk) characteristic of intracellular developing parasites versus the speckled UIS4 localization within the sporozoites (white arrow). In the quantification of (A), only parasites with UIS4 PVM staining were counted positive for UIS4. Anti-CSP antibody (green) was used to visualize al parasites (intracellular and extracellular). The nucleus was stained with DAPI (blue). (C) Schematic of PyROM4TMGFP construct displaying the truncated sequence of PyROM4 used to clone into a vector containing an IgK leader sequence, an N-terminal GFP tag (green hexagon), and a C-terminal 2xMyc tag (gray triangle). Dashed blue parallel lines represent the boundaries of the TMD (amino acid sequence in red font color) as predicted by HMMTOP and TMHMM analysis tools (Expasy.org). Arrow depicts the predicted rhomboid cleavage site between an Alanine and a Glycine. Predicted full length size for PyROM4TMGFP is 40 kDa, and the predicted size for the N-terminal rhomboid cleaved product is a 34 kDa. (D and E) Western blot analysis of transiently transfected COS7 cells used to detect proteolytic activity of rhomboid against PyUIS4TMGFP. (D) Western blot of cell lysates probed with anti-HA antibody (Cell α-HA) show proper expression of DmRho-1 (Dm), TgROM5 (TgR5), and TgROM5^SA (TgR5^SA) in the first three lanes and no detection of protein in the “no rhomboid” (–) negative control in the fourth lane. (E) Processing of PyUIS4TMGFP (anti-GFP_UIS4) was analyzed by western blot of cell lysates (upper panel, Cell) and concentrated media fraction (lower panel, Media) by probing with anti-GFP antibody. Full length PyUIS4GFP is observed in the cell fraction at the expect size of ∼40 kDa. A lower molecular weight product (∼34 kDa) is detected in both cell and media fractions (arrow) only in the first lane where DmRho-1 (Dm) is co-expressed. No cleaved product is seen when PyUIS4TMGFP is co-expressed with TgROM5 (TgR5), TgROM5^SA (TgR5^SA), and the “no rhomboid” negative control (–).