Scale bar?=?5 m

Scale bar?=?5 m.(1.45 MB TIF) ppat.1000309.s005.tif (1.3M) GUID:?756B84C9-5822-4E6D-9EEC-3411A13E8808 Physique S6: RON4, RON5, and RON8 remain associated with vacant parasitophorous vacuoles. were affinity-purified with the anti-RON5 antibody in 0.6% SDS conditions. Proteins that did not bind to the resin were collected (Flow Through: FT). (Right) Proteins isolated around the RON8 immunosorbents in 1% NP-40 conditions preserving the MJ complex were separated on SDS-PAGE, transferred to nitrocellulose, and probed SSH1 with secondary antibody conjugate alone (Conj), with anti-RON8, or with anti-RON5. Both fragments of RON5 (110 and 30 kDa) were also affinity-purified with anti-RON8 in 1% NP-40. Conj: alkaline phosphatase-conjugated secondary antibody control. Molecular masses are indicated.(0.85 MB TIF) ppat.1000309.s002.tif (834K) GUID:?0B853F83-8BFE-4554-963A-4483C62E363F Saxagliptin hydrate Physique S3: Saxagliptin hydrate Ty-tagging of RON4 leads to the accumulation of the non-mature form of the protein in the parasitophorous vacuole. (A) Intracellular parasites expressing Ty-tagged RON4 were processed for IFA after permeabilization with triton X-100 using anti-Ty to label the tagged protein of interest and anti-ROP1 to label the rhoptries. (B) Western blot analysis of cell extracts from control HX parasites or Ty-RON4-expressing transgenic parasites. Anti-RON4 monoclonal (left) labels mature and non-mature RON4 as well as the non-mature form of the Ty-tagged version of the protein in the transgenic cell line. Anti-Ty (right) specifically labels the tagged RON4 protein.(1.04 MB TIF) ppat.1000309.s003.tif (1012K) GUID:?C69CB395-717E-459C-8FDD-8F42466AE7C1 Physique S4: Antibody against RON8 pro-peptide specifically recognizes the non-mature form of RON8. (A) Immuno-precipitation after pulse-chase (P, C) metabolic labelling of RON8 in the absence or presence of BFA (to prevent maturation of RON8) shows that the anti-proRON8 antibody immuno-precipitates the non-mature form of RON8 specifically. surface protein SAG1) and positive (PVM-associated protein GRA3) controls were used to monitor the integrity of the PVM in the conditions used. Exposure of RON4, RON5, and RON8 to the outside of the vacuole was assessed using Anti-RON4 Mab, anti-RON5, and anti-RON8 antibodies. Scale bar?=?5 m.(1.45 MB TIF) ppat.1000309.s005.tif (1.3M) GUID:?756B84C9-5822-4E6D-9EEC-3411A13E8808 Figure S6: RON4, RON5, and RON8 remain associated with empty parasitophorous vacuoles. IFA on HFF cells pulse-invaded for 15 min, permeabilized with saponin, and incubated with anti-RON4, anti-RON5, and anti-RON8 antibodies, detected these RONs at a residual structure associated both with parasite-containing vacuoles (arrows) and vacant vacuoles (arrowheads). Scale bar?=?5 m.(2.31 MB TIF) ppat.1000309.s006.tif (2.1M) GUID:?15E79F12-E180-4AFA-9BE1-58E322899C78 Table S1: Peptides identified(0.03 MB DOC) ppat.1000309.s007.doc (30K) GUID:?F4B25414-D868-4B63-AC4F-6C4419924415 Text S1: Supplementary materials(0.07 MB DOC) ppat.1000309.s008.doc (73K) GUID:?09D6E43D-278E-4FA7-A4C1-841D45EB906C Abstract One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ Saxagliptin hydrate was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also exhibited that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms.