Next day, beads were washed three times with PBS, and the captured proteins were resolved on a 12% SDS-PAGE gel. Proteins were transferred into a nitrocellulose membrane and blocked overnight with Odyssey blocking
buffer (Li-Cor) in TBS (Tris-buffered saline). The membranes were probed with EEA1, buy SCH772984 CREB-1, MARCO and α-tubulin antibodies (Santa Cruz Biotechnology) for 1 h and after, incubated with appropriate secondary antibodies (Li-Cor) in TBS for 1 h. Proteins were visualized by scanning of the membranes in the Odyssey Imager (Li-Cor, Lincoln, NE). Concentration of single elements in the phagosome Human monocyte-derived macrophages were purified as previously described [17, 28], seeded on 200-mesh IDO inhibitor Formvar-coated London finder gold grids (Electron Microscopy Sciences) and cultured selleck products in RPMI-1640 supplemented with 10% FBS. The monolayers were infected with mycobacteria (MOI 10) for 1 h and subsequently washed with PBS. The monolayers were maintained in culture for 1 h or 24 h, then fixed and prepared for x-ray microscopy, as previously reported [17, 44], and the phagosome was obtained [17, 44, 45]. Elemental maps were extracted from x-ray fluorescence spectra, using the software package MAPS [47], and quantification was achieved by measuring x-ray fluorescence
from NIST thin-film standards NBS 1832 and NBS 1833 (National Bureau of Standards, Gaithersburg, MD, USA), prior to, during, and after the experiments. Calibration curves and calculations were carried out as described [17, 44, 45]. Statistical analysis of observed elemental changes was performed by comparing the concentration of the respective elements using Student’s-t test. A p < 0.05 was considered significant. Statistical analysis Comparisons between control and experimental groups were submitted to statistical analysis to determine the significance. Statistical analysis of the means ± SD was determined by ANOVA. A p < 0.05 was considered significant. A DNA microarray was carried out three independent times, while the proteomic analysis of vacuole proteins was performed twice. Acknowledgements We are grateful for the support of the Mass Spectrometry
Core Facility of the Environmental Health Sciences Center, Oregon State University, and from grant number P30 ES00210, from National Institute MycoClean Mycoplasma Removal Kit of Environmental Health Sciences, National Institutes of Health. This work was also supported by the NIH grants # AI47010 and AI043199. We thank Denny Weber for help in preparing the manuscript. References 1. Falkinham JO: Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 1996,9(2):177–215.PubMed 2. Inderlied CB, Kemper CA, Bermudez LE: The Mycobacterium avium complex. Clin Microbiol Rev 1993,6(3):266–310.PubMed 3. Aksamit TR: Mycobacterium avium complex pulmonary disease in patients with pre-existing lung disease. Clin Chest Med 2002,23(3):643–653.PubMedCrossRef 4.