The combination of these two obstacles renders the project challenging and explains why so few candidates were found in this work. Four of the identified proteins are enzymes of the glycolysis pathway (while Hexokinase is not specific to glycolysis). Germany) and was cultured according to Trager and Jensen  in human A+ erythrocytes and in a RPMI medium made up of neomycin, glutamine, Na2CO3 and human fresh frozen plasma (FFP) to reach a final haematocrit of 5% (v/v). The culture bottles were incubated in an environment made up of 5% (v/v) O2, 5% (v/v) BI-7273 CO2 and 90% (v/v) N2 at 37?C. The medium was changed every 1C2?days. The culture was microscopically monitored by Giemsa-stained smears. Contamination with Mycoplasma was ruled out by PCR control. The erythrocytes were separated by using SuperMACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in a magnetic field, hereby erythrocytes infected with late trophozoites were isolated from BI-7273 uninfected red blood cells and erythrocytes made up of parasites in the ring form in a magnetic field . The parasites were extracted by lysis of the erythrocytes membranes using a saponin buffer . Protein extraction The parasites were lysed on ice in the following buffer: 10?mM Tris/HCl, 150?mM NaCl, 1?mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, pH 7.4. After vigorous mixing and sonication, the lysate was centrifuged at 20,000for 10?min at 4?C. The pellet was discarded and the supernatant saved for further analyses. For incubation with PNGase F parasites were lysed in the following buffer: 10?mM Tris/HCl, 1?mM EDTA, 1?mM EGTA BI-7273 and 0.5% (v/v) Triton X-100, pH 7.5. For sWGA Rabbit Polyclonal to OR10H2 (succinylated-wheat germ agglutinin)-beads enrichment, a hypotonic buffer was used; the composition was as follows: 10?mM Tris/HCl, 1?mM MgCl2, 10?mM NaCl, 15?mM 2-mercaptoethanol, pH 7.2. A cocktail of proteases inhibitors was added to each buffer. SDS-PAGE and western blot Proteins BI-7273 were resolved on 8 or 10% SDS-PAGE and either brilliant blue stained or electroblotted onto nitrocellulose sheet. Equal loading and transfer efficiency were checked using Ponceau red staining. Membranes were saturated in 5% (w/v) non-fatty milk in Tris buffered Saline (TBS)-Tween [15?mM Tris, 140?mM NaCl, 0.05% (v/v) Tween, pH 8.0] for 1?h or in 5% BI-7273 (w/v) bovine serum albumin (BSA) in TBS-Tween overnight. The primary antibodies (anti-UDP-GlcNAc is the end-product of the hexosamine biosynthetic pathway (a). Among other GlcNAc transferases, UDP-GlcNAc is the substrate of OGT (adapted from Aquino-Gil et al. ). HK/GK, hexokinase/glucokinase; GPI, glucose-6-phosphate isomerase; GFAT, glutamine: fructose-6-phosphate amidotransferase; GNPNAT1, GlcNH2-6-phosphate and erythrocytes were run using SDSPAGE (right panel). The gel was stained with brilliant blue Mass spectrometry Specific bands were excised from brilliant blue stained gels. The pieces of gel were reduced using a 50:50 dilution of 50?mM ammonium bicarbonate (Bic) (HPLC Grade, Prolabo) and acetonitrile (ACN) (Sigma A) followed by 100% (v/v) ACN. They were then reduced in 20?mM DTT (Sigma) in 50?mM Bic and alkylated in 100?mM iodoacetamide (Bio-Rad) in 50?mM Bic. After washing in ACN/Bic the bands were digested with 100C200?ng trypsin gold (Promega) in 25?mM Bic. Extraction was done using 45% (v/v) ACN and 10% (v/v) formic acid (Sigma). The extracted peptides were purified using C18-Zip-Tip cones using 0.1% (v/v) formic acid for washing and 50:50 ACN/0.1% (v/v) formic acid to elute the purified sample. The nano-LC MS/MS analysis was performed on a HPLC system with two LC-20AD nano-flow LC pumps, a SIL-20 AC auto-sampler and a LC-20AB micro-flow LC pump (Shimadzu, Kyoto, Japan) connected to an ion-trap mass spectrometer (amaZon ETD, Bruker Daltonics, Bremen, Germany) equipped with a Captive Spray ion source. Hystar (Version 3.2, Bruker Daltonics, Bremen, Germany) was used to couple and control Shimadzu CBM-20A module (Shimadzu, Kyoto, Japan) for MS acquisition for all those experiments. Trapping and desalting of the peptides was carried out on nano trapping column (Zorbax 300SB-C18, 5?m, 0.3??5?mm, Agilent) using 0.05% (v/v) trifluoroacetic acid solution for 10?min at a flow rate of 10 L/min. After back-flushing from the trapping column, peptides were separated on a reversed-phase column, ACQUITY UPLC? M-Class Peptide BEH C18 Column (1.7?m, 130 ?, 100??0.75?mm i.d., Waters) using an acetonitrile/0.1% (v/v) formic acid.