The percentage of 15N in the labeled media is more than 98% (Silantes GmbH, München, Germany). The cultures were inoculated with a starter culture grown in normal (14N) or 15N-labeled media until

mid-log phase. Two hundred fifty milliliter culture medium was inoculated with each starter Lorlatinib manufacturer culture and grown at 37°C with shaking at 225 rpm for 4 h. 15N-labeled culture was treated with 5 mM H2O2, which is well below the minimal inhibition concentration (MIC) of SE2472 (20 mM), and both cultures were grown for 2 h following the addition of H2O2. Protein extraction was performed with B-PER® bacterial protein extraction reagent (Thermo Fisher Scientific, Rockford, IL) and quantified with Dc Protein Assay Kit (Bio-Rad, Hercules, CA), which has an error rate selleck screening library of 2.5% in our experiments. We took this error rate into consideration by classifying any protein that had a 5% change or less as unchanged (having a 0% change). Two-dimensional gel electrophoresis and visualization of bacterial

proteins Protein samples were further solubilized in rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte® 3/10 ampholytes [Bio-Rad, Hercules, CA] and trace amount of Bromophenol Blue). ReadyStrip™ IPG strips (Bio-Rad, Hercules, CA) were loaded with 200 μg of protein samples (either normal or 1:1 mixture of normal and 15N-labeled samples) for preparative 2 D gels, and allowed to rehydrate for 18-22 h. Isoelectric focusing (IEF) was performed at 20°C using PROTEAN® IEF cell (Bio-Rad, Hercules,

CA). A 3-step protocol (250 V-20 min/8,000 V-2.5 h/8,000 V-10,000 V.h) was used for the IEF procedure following manufacturer’s recommendations (Bio-Rad, Hercules, CA). After the IEF procedure, the IPG strips were reduced in Equilibration Buffer I (6 M urea, 2% SDS, Anacetrapib 0.375 M Tris-HCl [pH 8.8], 20% glycerol, 2% DTT) and alkylated in Equilibration Buffer II (6 M urea, 2% SDS, 0.375 M Tris-HCl [pH 8.8], 20% glycerol, 0.25% iodoacetamide). Strips were loaded onto 8-16% Criterion™ Tris-HCl SDS gel (Bio-Rad, Hercules, CA) and electrophoresed at 200 V for 65 min. Gels were visualized using Coomassie Brilliant Blue R-250 or silver staining (Invitrogen, Carlsbad, CA). Mass spectrometric identification of proteins Gels were scanned and protein spots of interest were excised using the Xcise automated gel processor (Proteome Systems, North Ryde, Australia). Gel spots were destained and washed, followed by in-gel tryptic digestion using proteomic grade trypsin (Sigma-Aldrich, St. Louis, MO). Peptide fragments were collected and purified using ZipTip™ C18 reverse-phase prepacked resin (Millipore, Billerica, MA) and mixed with an equal volume of 10 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid (Sigma-Aldrich, St. Louis, MO) in 0.1% trifluoroacetic acid (TFA)/50% acetonitrile solution and directly spotted onto a stainless steel target plate for mass analysis.