N-Chlorotaurine Inacitvates Shiga Toxin Figure 7. Survey of the impacts of NCT on Stx2. The found effects of NCT on Stx2 are summarized in this figure. doi:10.1371/journal.pone.0047105.g007 the manufacturers’ protocol for FACS. Cell viability was checked with propidium iodide assay (Sigma-Aldrich). Cells were prepared in standard procedure for FACS analysis. Measurement of 5.000 events out of 150.000 cells per sample was carried out by BD FACS Canto II. Mouse IgG1:FITC (GM4992), ordered from invitrogen, was used as isotype control. Results were given as ratio of mean fluorescence intensity (MFI) of sample and MFI of isotype to avoid biases due to background fluorescence. Penetration of NCT-treated Stx2 into GEnC (Confocal Laser Scanning Microscopy) Labeling of Stx2 was done with OysterH-488 Antibody Labeling Kit (Luminartis, Muenster, Germany) as described by the manufacturer. Subsequently, labeled Stx2 was incubated in 55 mM NCT (1% w/v) in PBS (or plain PBS for controls) for 30 min at 37uC. Then, an aliquot of 2 ml of this mixture was added to the cells in 200 ml cell culture medium to reveal a final concentration of 20 pg/ml of labeled Stx2 and to reduce the concentration of NCT below cytotoxic levels. Immediate confocal laser scanning microscopy to visualize the intracellular accumu- lation of labeled Stx2 was performed on a microlens-enhanced Nipkow disk-based confocal system UltraVIEW RS (Perkin Elmer, Wellesley, USA) mounted on an inverted microscope (IX-70, Olympus, Nagano, Japan). Images were taken using the Ultra- VIEW RS software (Perkin Elmer) in 6000x magnification with oil. To exclude destruction of OysterH 488-TFP by NCT, both components were mixed at the same concentrations and conditions as for confocal laser scanning microscopy. Immediately afterwards, 10 ml were pipetted onto an object slide without additives and monitored by fluorescence microscopy over a period of 30 min. In addition, 1 ml of OysterH 488-TFP was 100-fold diluted with 10% w/v lysine in water followed by a 10-fold dilution in 1% w/v NCT or saline control. The fluorescence signal was only minimally decreased compared to the fluorescent dye without NCT in both settings, so that the tests were not biased by a reaction of NCT with the dye. Structural Changes of Stx2 by NCT Evaluated by SDS- polyacrylamid Gel Electrophoresis (SDS-PAGE) An electrophoresis system from Biorad was used to perform SDS-PAGE with a 16% gel. To exceed the minimum of 5mg protein required for mass spectrometry, we used 10 mg and 15 mg Stx2 per slot. To achieve these quantities of Stx2, 4 ml purified Stx2 (5 mg/ml) were mixed with 20 ml 1.2% w/v NCT (dissolved in phosphate buffer, pH 7.1), and additionally 6 ml purified Stx2 with 18 ml 1.2% w/v NCT, and incubated for 30 min at RT. A control was performed in parallel without NCT. The samples were bisected into 12 ml each and mixed with 3 ml reducing or non- reducing buffer each. The sample buffer consisted of 250 ml 20% w/v SDS, 500 ml 0.5 M Tris buffer, 200 ml glycerol and 100 ml 1% w/v bromphenol blue solution.The reducing buffer contained 50 ml mercaptoethanol as a reducing substance (final concentra- tion 4.5% w/v). Subsequently all samples were boiled at 95uC for 5 min and placed on ice immediately afterwards. For improved separation of low molecular-weight proteins, particularly for Stx2 subunit B, we applied a 16.5% tricine gel containing 6M urea, as described by Scha¨gger [42]. Gels were stained with coomassie blue. For mass spectrometry, only bands from samples in non-reducing buffer (without mercaptoethanol) were cut out to detect oxidations of thiols. Structural Changes of Stx2 by NCT Evaluated by Mass Spectrometry The in-gel digestion was performed as published previously [43]. Protein digests were analyzed using an UltiMate 3000 nano- HPLC system (Dionex, Germering, Germany) coupled to an LTQ Orbitrap XL mass spectrometer (ThermoScientific, Bremen, Germany) equipped with a nanospray ionization source. A homemade fritless fused silica nano-capillary column (75 mm i.d. 6 280 mm o.d.) packed with 10 cm of 3 mm reversed-phase C18 PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e47105