And 0.05 glutaraldehyde in PBS (2 min), neutralized by 0.1 M glycine/PBS (five min), after which blocked in 3 BSA/PBS (30 min). Noninvasive parasites or egressed vacuoles have been stained with antiTgSag1 antibody (1:1,500, 1 hr) before 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 had been washed and immunostained with Alexa488 and Alexa594conjugated antibodies (1:three,000, 1 hr). The number of invaded parasites was deduced by immunostaining with antiTgGap45/Alexa594 (red), but not with antiTgSag1/Alexa488 (green). The egressed vacuoles were scored directly in the number of vacuoles with TgSag1stained parasites.4-Formylaminoantipyrine Protocol Immunofluorescence LocalizationLocalization of epitopetagged proteins was performed by immunofluorescence assays. The process was essentially 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 employed together with antiHA antibody (1:five,000; SigmaAldrich, Germany) to assess localizations of epitopetagged PSS and PTS proteins. Abscisic acid Technical Information Photos have been acquired working with 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 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 were 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 Needed for the Parasite VirulenceLipid Extraction, TLC, and Phosphorus QuantificationParasites have been syringereleased from infected HFF (MOI, three; 428 hrs of infection) and passed twice by way of 23G and 27G needles. Host debris was removed by filtering the parasite suspension by means of a 5 m filter (Merck Millipore, Germany). Cell pellets (0.51×108 parasites) have been resuspended in 0.4 ml of PBS and lipids have been extracted as outlined by BlighDyer [41]. Briefly, 0.five ml chloroform and 1 ml methanol were mixed for the samples, which have been permitted to stand for 30 min and centrifuged (2,000 g, five 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 and also the decrease chloroform phase containing lipids was transferred to a conical glass tube. Samples were stored at 20 within the airtight glass tubes flushed with nitrogen gas. Lipids have been resolved by twodimensional TLC on silica gel 60 plates (Merck) making use of chloroform/methanol/ ammonium hydroxide (65:35:five) and chloroform/acetic acid/methanol/water (75:25:5:two.2) because the solvents for the initial and second dimensions, respectively. They have been visualized by staining with iodine vapors and identified based on their migration with genuine standards (Avanti Lipids). The big iodinestained phospholipid bands were scraped off the silica plate, and quantif.
