Triple primer PCR was performed with primer pairs repre- senting the genomic sequences flanking the PB gene trap insertion and one PB end primer (Supplementary table S1). The PCR reaction was competitive between the amplifications from the genomic sequences and the PB primer and genomic primer. The wild type, heterozygous or homozygous ES cell clone have different but comparable size PCR products. PBase reversal Five million cells were electroporated with 20 mg PiggyBac transposase (PBase) expression plasmid, plated on 90 mm feeder plates, and 2 days later the cells were re-plated at low density (1,000 cells/10 cm plate) to allow the growth of individual ES cell colonies. Clones were picked into 96-well plates and the deletion of the PB transposon was confirmed by PCR. Ricin resistance was assessed by plating105 cells/well in 24-well plates, exposing to ricin for 3 days and 6 days further culture with normal medium. Cells were visualized by staining with 2% methylene blue as described previously. Lectin screen Kits of fluorescein isothiocyanate (FITC) conjugated lectins, encompassing several sugar specificities, were purchased from Vector Laboratories. Cells were harvested by trypsinisation and re-suspended at 16106/ml in staining buffer (16 Dulbecco’s (D)- PBS containing 5% foetal calf serum and 0.01% sodium azide). Aliquots of 100 ml were incubated with 2 ml (1–2 mg/ml) lectin for 20 m at 4uC prior to extensive washing with D-PBS. After fixation in 100 ml 1% paraformaldehyde in D-PBS, stained cells were analysed by flow cytometry on the FL-1 green channel of a Becton Dickinson FACsAria. Glycosylation inhibitors The inhibitors deoxynojirimycin, deoxymannojirimycin and swainsonine (VWR) were diluted to 100 mM in sterile D-PBS as stock solutions. Further dilutions of 1, 10 and 100 mM [15] were prepared in either normal media, for pre-treatment, or in medium containing 10 pM ricin. AB2.2 or NN5 cells, plated at 0.56105/ well in 24 well plates one day earlier, were pre-treated, in duplicates, with inhibitor for initially 4 h at 37uC prior to ricin exposure. The inhibitor was then removed and replaced with fresh medium containing either inhibitor and/or 10 pM ricin or ricin alone. The cells were exposed for 3 days before neutral red staining. Due to the nature of ES cell growth in that domed colonies form and not monolayers, pre-treatment was extended to 24 hours in subsequent experiments to ensure full exposure to the inhibitors. Glycomics analysis Pellets of 26106 cells were washed once in PBS prior to freezing at 280uC. Corresponding aliquots of the relevant mediums were also frozen at the same time. Glycoproteins and glycosphigolipids (GSLs) were extracted and separated by homogenisation and solvent partition ([29]. Cells were homogenised in 4 volumes of ice-cold water, the total aqueous volume was determined and glycolipids were extracted from the homogenate, by addition of 2.67 volumes of methanol and 1.33 volumes of chloroform, and vortexed thoroughly before centrifugation at 3000 rpm for 10 min. The glycoproteins were pelleted, reduced and carbox- ymethylated. After dialysis and lyophilisation, samples were digested with trypsin (Bovine TPCK treated T-1246, Sigma, Poole, UK) as previously described [30] and glycopeptides/ peptides were purified on SepPak cartridge C18 (Waters Corp, Man2, a1 and Ricin Resistance in ES Cells Elstree, UK) [30]. The glycopeptides/peptides were dissolved in 200 ml of Ambic buffer (50 mM, pH 8.4). 3 U of PNGase F (1365177, Roche Applied Science, Burgess Hill, UK) was added and the reaction was incubated at 37uC for 24 hr, adding a fresh 3 U aliquot of enzyme after 12 hr. The digested samples were lyophilised and redissolved in 200 ml of 5% (v/v) acetic acid before being loaded onto a preconditioned Classic C18 Sep-PakH and eluted with 5 ml of 5% (v/v) acetic acid (the N-glycan fraction) followed by elution with 4 ml of 20% and 40% (v/v) propan-1-ol in 5% (v/v) acetic acid (peptide/O-linked glycopeptide fractions). The 5% (v/v) acetic acid and combined 20% and 40% (v/v) propanol fractions were lyophilised. Permethylation and sample clean-up were performed using the sodium hydroxide protocol, as described previously [30]. Combined 20% and 40% (v/v) propanol fractions (peptide/O-linked glycopeptide fractions) resulting from the post-PNGase F digest were dissolved in 800 ml of 1 M potassium borohydride (KBH4) in a 0.1 M solution of potassium hydroxide (KOH) and incubated at 45uC for 20–22 hr. The reaction was terminated by drop wise addition of glacial acetic acid (5 drops). Cationic salts, amino acids and peptides were removed from the sample using a Dowex (50W-X8 (H+) 50– 100 mesh) column. The O-glycans fractions obtained were then permethylated using the same method as the one used for the N- glycans. After extraction of the pellet of glycoproteins, the supernatant containing the GSLs was transferred to a fresh tube and water (0.173 volumes of supernatant) added. After mixing and centrifugation (15506 g, 10 min), two phases were obtained. The lower phase was extracted twice with water and dried under nitrogen. The upper phase was cleaned-up on a C18 Plus Sep-Pak cartridge (Waters, Elstree, UK) as described earlier [29]. GSLs were incubated with ceramide glycanase (rEGCase II, 4460, Takara, Shiga, Japan). The reaction mixture was extracted with 1- butanol and the aqueous phase cleaned-up on a C18 Sep-Pak column. The glycans in the unbound fraction were purified on Hypercarb Cartridges (Waters, Elstree, UK) and freeze-dried. Glycans were deuteroreduced by 10 mg/mL sodium borodeuter- ide in 2 M ammonia solution (2 h, 25uC). After neutralization with acetic acid, the borates were removed by co-evaporation with 10% (v/v) acetic acid in methanol. Permethylation of glycans was performed using the sodium hydroxide procedure [31]. MALDI-TOF MS and MALDI-TOF/TOF MS/MS data were acquired using a 4800 MALDI TOF/TOF (Applied Biosystems) mass spectrometer. The MS spectra were obtained using the reflector positive mode with delayed extraction. MS/MS data were acquired using collision energy of 1 kV, and argon was used as collision gas. Samples were dissolved in 10 ml of methanol and mixed at a 1:1 ratio (v/v) with 2,5-dihydrobenzoic acid as matrix. Immunogold staining of ricin and transmission electron microscopy Cells incubated with 20 pM ricin for 1 h were fixed with 2% paraformaldehyde/0.2% glutaraldehyde in PBS, at 37uC for 10 minutes and then transferred to ice. The cells were rinsed three times with PBS and briefly quenched with 0.1% glycine in PBS at 37uC, covered with 1 ml of 1% gelatin, scraped with a freshly cut Teflon strip into a 1.5 ml Eppendorf and centrifuged at 3,000 rpm. The resulting pellet was gently re-suspended in 10% gelatin and centrifuged again before chilling at 4uC. Small chopped pieces were infiltrated with 2.3 M sucrose on a roller at 4uC overnight and then mounted on aluminium stubs and plunge frozen in liquid nitrogen. 60 nm ultrathin cryosections were cut on a Leica EM FC6 ultra microtome and labelled with rabbit anti- ricin toxin A chain (Abcam) followed by 10 nm protein A gold PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e22993