br Data After growing Salmonella Typhimurium bacteria under

1. Data After growing Salmonella Typhimurium bacteria under a variety of environmental conditions, bacterial survival was determined after exposure to oxidative stress. Secondly, the production of endogenous hydrogen peroxide was measured of bacteria that were grown to different growth phases. Lastly, bacterial survival of S. Typhimurium was analyzed after exposure to different antibiotics.
2. Experimental design, materials and methods
Acknowledgments This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR-27R18706). B.B.F. is the University of British Columbia Peter Wall Distinguished Professor. No competing interests exist for this work.
Data The data is related to the identification and verification of transgelin-2 as a potential biomarker of tumor-derived lung-cancer endothelial Phenyl sulfate by comparative proteomics [1]. A highly optimized method for primary CD105+ NECs and TECs proteomic analysis by 2D-PAGE and MALDI-MS/MS was presented here. All the identified proteins were categorized by GO, KEGG and protein–protein interaction analysis, to clarify the function of TEC in tumor microenvironment.
Experiment design, materials and methods Primary CD105+ NECs and TECs were isolated from a mouse Lewis lung carcinoma model bearing 0.5cm tumors. Differentially expressed proteins were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). 2D-PAGE was performed using the GE Ettan™ IPGphor™ 3 and DALTsix system. Proteins were visualized by silver staining, and images were recorded on a GE ImageScanner III system and analyzed with the ImageMaster 2D Platinum software. Mass spectrometry data were obtained in an automated analysis loop using a 4800 Plus MALDI TOF/TOF™ Analyzer (Applied Biosystems, USA), and collected using the 4000 Series Explorer™ software and submitted to database search via GPS Explorer™ (Applied Biosystems). MASCOT Server version 2.2 and NCBI non-redundant database were used for protein identification. A total of 63 spots resulted in the identification of 48 unique proteins (28 up- and 20 down-regulated proteins) were detected by at least 1.5-fold changes in TECs. All the identified proteins were categorized functionally by Gene Ontology (GO) analysis. Gene-pathway annotations were compiled from Kyoto Encyclopedia of Genes and Genomes (KEGG), BioCarta, BioCyc, and Reactome. Protein–protein interaction networks were built using the DIP, MINI, BioGRID, IntAct, and STRING databases, and the data were imported into Cytoscape in order to visualize the graphs.
Establishment of ECs cultures Primary ECs were purified by combining the enzymatic digestion, differential adherence and magnetic cell-sorting using a CD105 MultiSort Kit, according to the procedure described in the Journal of Proteomics paper [1]. Endothelial phenotype and purity were confirmed by cytofluorimetric analysis on the basis of positive expression of a panel of endothelial markers [2,3], and isotype control stainings were shown in Fig. 1. ECs (CD105 expression of >98%) at first passage were used for the proteomic analysis to maintain the most properties of the in vivo state [4].
Comparative proteomic analysis of NECs and TECs NECs and TECs were harvested and suspended in lysis buffer containing 7M urea, 4% CHAPS, 2M thiourea, 60mM DTT, 10mM Tris, 1mM EDTA, 0.002% bromophenol blue, and 2% ampholine (pH 3–10) [5]. Cells were disrupted on ice by five 15s pulses of sonication, followed by five cycles of freeze-thaw: 5min in liquid nitrogen, 1min in a 37°C water bath and 3min at room temperature. Then, supernatant fractions were collected after centrifugation at 14,000×g for 40min at 4°C and then stored at −80°C. Protein concentration was determined using a Bradford assay kit. A non-linear pH gradient of 3–10 was chosen for isoelectric focusing (IEF). The second-dimension was performed on a 12.5% SDS-PAGE to optimize the separation of proteins from 12 to 97kDa. Before IEF, a solution containing 50mM MgCl2, 1mg/mL DNase 1, and 0.25mg/mL RNase A was added to the protein samples at ratio of 1:20 (V/V). Aliquots containing 100μg of protein were resuspended in 250μL of rehydration solution. Equal amount of sample was loaded in triplicate. After 18h of rehydration of the IPG strips (GE Healthcare, USA), IEF was performed using the GE Ettan™ IPGphor™ 3 system at 67,860V·h. After focusing, the strips were first equilibrated for 15min in a buffer containing 6M urea, 20% glycerol, 2%SDS, 2% DTT, and then for 15min in the same buffer containing 2.5% iodoacetamide instead of DTT. SDS-PAGE was performed on a GE Ettan™ DALTsix system. Finally, the proteins were visualized by silver staining. Briefly, gels were soaked in fix solution (50% ethanol, 10% acetic acid) for at least 45min, rinsed in 30% ethanol and ddw for 3×10min, respectively. To sensitize, gels were soaked in sensitivity enhancing solution (2mL of 10% sodium thiosulfate solution per liter) for 2min (one gel at a time), followed by rinsed in ddw for 2×1min. For silver reaction, submerged gel in 0.1% silver stain solution [0.1% silver nitrate with 0.08% formalin (37%)] for 20min, followed by rinsed in ddw for 2×1min. Developed image in development solution [2% sodium carbonate with 0.04% formalin (37%)] until desired intensity of staining occur, then quickly washed in 5% acetic acid for 10min, and rinsed in ddw for 5min to stop the staining. Finally, all gels were rinsed with water (several changes) prior to drying or densitometry.