And 0.05 glutaraldehyde in PBS (2 min), neutralized by 0.1 M glycine/PBS (five min), and then blocked in three BSA/PBS (30 min). Noninvasive parasites or egressed vacuoles were stained with antiTgSag1 antibody (1:1,500, 1 hr) prior to detergent permeabilization. Cells were washed 3x with PBS, permeabilized with 0.2 triton X 100/PBS (20 min), and stained with antiTgGap45 antibody (1:3,000, 1 hr) to visualize intracellular parasites. Samples have been washed and immunostained with Alexa488 and Alexa594conjugated antibodies (1:3,000, 1 hr). The amount of invaded parasites was deduced by immunostaining with antiTgGap45/Alexa594 (red), but not with antiTgSag1/Alexa488 (green). The egressed vacuoles have been scored straight from the number of vacuoles with TgSag1stained parasites.Immunofluorescence LocalizationLocalization of epitopetagged proteins was performed by immunofluorescence assays. The approach was primarily the exact same as described for invasion assays except for that samples have been permeabilized prior to incubation with antibodies. A panel of organellespecific antibodies (TgMic2 for micronemes, 1:1,000; TgRop1 for rhoptries, 1:1,000; TgGra5 for dense granules, 1:500; TgF1B for mitochondrion, 1:1,000; TgFd for apicoplast, 1:500; TgVP1 for acidocalcisomes/plantlike vacuole, 1:500) was used with each other with antiHA antibody (1:five,000; SigmaAldrich, Germany) to assess localizations of epitopetagged PSS and PTS proteins. Images have been acquired using ApoTome microscope (Zeiss, Germany).Functional Expression in E. coliThe M15/pREP4 strain was transformed together with the empty pQE60 expression vector (Qiagen), pQE60TgPTS, pQE60TgPSS, or pQE60AtPSS [17] constructs and cultured in LuriaBroth medium supplied with ampicillin (one hundred mg/L) and POM1 manufacturer kanamycin (50 mg/L). Protein expression was induced by 1 mM IPTG at 25 in overnight cultures containing 5 mM threonine or serine, followed by a four hr incubation at 37 . Lipids had been isolated and separated by onedimensional TLC in chloroform/methanol/acetate (130:50:20) and visualized by ninhydrin staining.PLOS Biology | DOI:ten.1371/journal.pbio.November 13,17 /Phosphatidylthreonine Is Essential for the Parasite VirulenceLipid Extraction, TLC, and Phosphorus QuantificationParasites were syringereleased from infected HFF (MOI, 3; 428 hrs of infection) and passed twice via 23G and 27G needles. Host debris was removed by filtering the parasite suspension by way of a 5 m filter (Merck Millipore, Germany). Cell pellets (0.51×108 parasites) were resuspended in 0.4 ml of PBS and lipids had been extracted in accordance with BlighDyer [41]. Briefly, 0.5 ml chloroform and 1 ml methanol have been mixed to the samples, which were permitted to stand for 30 min and centrifuged (2,000 g, 5 min). The supernatant was transferred to a glass tube followed by addition of chloroform and 0.9 KCl (1 ml every single). Samples had been mixed, centrifuged as well as the reduced chloroform phase containing lipids was transferred to a conical glass tube. Samples have been stored at 20 within the airtight glass tubes flushed with nitrogen gas. Lipids had been resolved by twodimensional TLC on silica gel 60 plates (Merck) making use of chloroform/methanol/ ammonium hydroxide (65:35:5) and chloroform/acetic acid/methanol/water (75:25:five:2.2) because the solvents for the first and second dimensions, respectively. They had been visualized by staining with iodine vapors and identified according to their migration with authentic requirements (Avanti Lipids). The main iodinestained phospholipid bands had been scraped off the silica plate, and quantif.
