Representative images for segmentation and quantification of GFP-TSG101 to the PVM between WT, Rand Rcomplement strains. analysis of the internalization of host cytosolic proteins of TgGRA14-deficient parasites. (1) Parasites were treated with 5 M LHVS for 24 h prior to contamination, (2) inducible mCherry HeLa cells were infected with parasites for 4 h, (3) the parasites were harvested at 4 hpi and analyzed by microscopy. B. Quantification of host cytosolic mCherry uptake at 4 hpi by WT or Rtype I strains treated with Zabofloxacin hydrochloride DMSO or LHVS for 24 h. At least 200 parasites were analyzed per blinded sample. Data represents the mean from 3 biological replicates. Statistical analysis was by Students t-test. Only statistical differences are shown.(DOCX) ppat.1010138.s003.docx (314K) GUID:?61C355EA-F91D-41B5-AF67-3093578B6B1D S4 Fig: Deletion of TgGRA14 in type II strain. A. Schematic for the deletion of the HXGPRT gene with primer binding sites B. PCR to validate the deletion of HXGPRT. C. Schematic for the deletion of the TgGRA14 with primer amplification sites. D. PCR to validate the deletion of TgGRA14 in the ME49background. E. Immunoblot confirming the deletion of TgGRA14 in the Mstrain.(DOCX) ppat.1010138.s004.docx (395K) GUID:?5639AB22-363B-451A-96CE-A1AB7F64C34A S5 Fig: Rcomplementation mutants. A. Schematic representation of the plasmids generated for TgGRA14 Zabofloxacin hydrochloride complementation. B. PCR to validate the presence of TgGRA14-HA in the Rcomplementation mutants. C. Immunoblot confirming the presence of TgGRA4 and HA in the Rcomplementation mutants. D. Representative images showing the presence of HA and GRA14 in the Rcomplementation mutants. Scale bar is usually 5 m.(DOCX) ppat.1010138.s005.docx (812K) GUID:?0305645B-6840-44E3-A8F2-0EE75E64EDCF S6 Fig: Topology of TgGRA14 late Zabofloxacin hydrochloride domain motif mutants at the PVM. A. Semi-permeabilization with 0.1% saponin for permeabilization of the host plasma membrane and PVM. B. Semi-permeabilization using 0.00001% saponin to only permeabilize the host plasma membrane and not the PVM. Detection of TgGRA14 N-terminus and C-terminus by probing against GRA14N and HA respectively. Scale bar is usually 5m.(DOCX) ppat.1010138.s006.docx (1.0M) GUID:?AB28ADFD-BA02-43A0-8CA4-3E51CE8E67B4 S7 Fig: Comparison of GFP-TSG101 recruitment to the PVM following disruption of late domain motifs. A. Representative images for segmentation and quantification of GFP-TSG101 to the PVM between WT, Rand Rcomplement strains. The PV was labeled using an antibody against TgGRA1. Level bar is usually 5 m. B. Replication analysis of TgGRA14 match strains in GFP-TSG101 HeLa cells by analyzing parasites (nuclei) per PV (labeled with GRA1). C. Measurement of growth by analyzing the PV size of TgGRA14 match strains in GFP-TSG101 HeLa cells. Data represents the mean from 3 biological replicates. Statistical analysis was by Kruskal-Wallis test. No statistical differences were detected between samples.(DOCX) ppat.1010138.s007.docx (1.0M) GUID:?8827755C-0078-4718-9D79-1DF9BDE67A20 S1 Dataset: LC-MS/MS Analysis. Data are offered in 9 different tabs. The Results Summary tab provides an overview and warmth map of the top protein hits identified for Rabbit polyclonal to ASH1 each of the three different conditions (Tz-HFF: tachyzoite fibroblast contamination; Bz-HFF: bradyzoite fibroblast contamination; Neuron: neuron contamination). The tabs labeled Condition_PeptideCalc demonstrate the analysis pipeline, including equations used in Excel to ultimately calculate log2 protein fold changes and p-values. The tabs labeled Condition_VolcanoPlot provide a graphical overview of protein fold changes and short names for the top protein hits. Lastly, the Peptide_RawData tabs provide identification scores and PEP Zabofloxacin hydrochloride values for each unique peptide recognized from either Toxoplasma-Human or Toxoplasma-Mouse combined proteome database searches.(XLSX) ppat.1010138.s008.xlsx (4.4M) GUID:?50648E3B-434C-4892-82FB-EC44EEE0BE6D S1 Table: proteins encoding a P(S/T)AP motif identified bioinformatically. Identification of effector proteins encoding a putative P(S/T)AP motif and a signal peptide as potential candidate interactors with the host ESCRT-I component, TSG101. Bioinformatic search was performed using the vEuPathDB Informatics Resources Database (ToxoDB) and their predicted localization is based on the hyperplexed localization of organelle proteins by isotype tagging (hyperLOPIT) data set [36].(DOCX) ppat.1010138.s009.docx (28K) GUID:?8CBA651F-6764-4DDA-A1BE-8E97DACF1EE2 Zabofloxacin hydrochloride S2 Table: Host and parasite proteins significantly enriched following immunoprecipitation with TgGRA14-HA. Immunoprecipitation of TgGRA14 with host proteins. List of TgGRA14 host-interacting proteins and interacting proteins in Tz-HFF samples with 1 log2 fold switch in tagged vs control lysates.