Western blot - mBiombio.asm.org/.../mbo005121342s1.docx · Web viewAfter 24 h confluent cells were...
5
Supplementary Material and Methods Inhibitors The PI3K LY294002 (New England Biolabs) inhibitor was used at a final concentration of 10 μM (added 30 min before the bacteria) Propidium iodide incorporation HeLa cells were seeded in 2-well chamber slides. After 48 h, cells were infected with primed bacteria for 30 min. Cells were washed 5 times with PBS and incubated in 2.5 µg/ml of propidium iodide (Invitrogen), PI, in DMEM without dye. Incorporation of PI was then monitored during the next 30 min by live cell epifluorescence microscopy using a Zeiss Axioimager microscope. Plasma membrane purification Plasma membrane purification was obtained using Qproteome Plasma Membrane Protein Kit (Quiagen). Briefly, cells were seeded in T75 cm flask and transfected the following day. After 24 h confluent cells were harvested and washed with PBS 3 times before processed according to the manufacturer’s recommendations. Plasma membrane fraction was precipitated using 4 volumes of ice cold acetone.
Transcript of Western blot - mBiombio.asm.org/.../mbo005121342s1.docx · Web viewAfter 24 h confluent cells were...
Supplementary Material and Methods
Inhibitors
The PI3K LY294002 (New England Biolabs) inhibitor was used at a final
concentration of 10 μM (added 30 min before the bacteria)
Propidium iodide incorporation
HeLa cells were seeded in 2-well chamber slides. After 48 h, cells were infected with
primed bacteria for 30 min. Cells were washed 5 times with PBS and incubated in 2.5
µg/ml of propidium iodide (Invitrogen), PI, in DMEM without dye. Incorporation of
PI was then monitored during the next 30 min by live cell epifluorescence microscopy
using a Zeiss Axioimager microscope.
Plasma membrane purification
Plasma membrane purification was obtained using Qproteome Plasma Membrane
Protein Kit (Quiagen). Briefly, cells were seeded in T75 cm flask and transfected the
following day. After 24 h confluent cells were harvested and washed with PBS 3
times before processed according to the manufacturer’s recommendations. Plasma
membrane fraction was precipitated using 4 volumes of ice cold acetone. After 15 min
of incubation on ice, pellet were collected by centrifugation, dry and resuspended in
200 µl of water.
Western blot
Proteins were resolved by SDS–PAGE, and gels were transferred to polyvinylidene
difluoride (PVDF) membrane (GE Healthcare). Membranes were washed with PBS
0.1% Tween, blocked in TBS (0.1% Tween, 3% BSA, 0.5% gelatin) and probed with
specific antibodies overnight. Blots were then incubated with horseradish peroxidase-
linked secondary antibody (Jackson ImmunoResearch), followed by ECL plus assay,
according to the manufacturer’s instructions (GE Healthcare). Chemiluminescences
were detecting using a LAS 3000 Fugi imager. Polyclonal anti-GADPH (Abcam),
monoclonal anti-Cytochrome-c (Merck) and monoclonal anti-myc (Millipore) were
used to detect the different proteins.
Supplementary References
1. Levine, M. M., E. J. Bergquist, D. R. Nalin, D. H. Waterman, R. B. Hornick, C. R. Young, and S. Sotman. 1978. Escherichia coli strains that cause diarrhoea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet 1:1119-1122.
2. Nadler, C., Y. Shifrin, S. Nov, S. Kobi, and I. Rosenshine. 2006. Characterization of enteropathogenic Escherichia coli mutants that fail to disrupt host cell spreading and attachment to substratum. Infect. Immun. 74:839-849.
3. Marches, O., S. Wiles, F. Dziva, R. M. La Ragione, S. Schuller, A. Best, A. D. Phillips, E. L. Hartland, M. J. Woodward, M. P. Stevens, and G. Frankel. 2005. Characterization of two non-locus of enterocyte effacement-encoded type III-translocated effectors, NleC and NleD, in attaching and effacing pathogens. Infect. Immun. 73:8411-8417.
4. Tzipori, S., H. Karch, K. I. Wachsmuth, R. M. Robins-Browne, A. D. O'Brien, H. Lior, M. L. Cohen, J. Smithers, and M. M. Levine. 1987. Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets. Infect. Immun. 55:3117-3125.
5. O'Brien, A. O., T. A. Lively, M. E. Chen, S. W. Rothman, and S. B. Formal. 1983. Escherichia coli O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella dysenteriae 1 (SHIGA) like cytotoxin. Lancet 1:702.
6. Riley, L. W., L. N. Junio, L. B. Libaek, and G. K. Schoolnik. 1987. Plasmid-encoded expression of lipopolysaccharide O-antigenic polysaccharide in enteropathogenic Escherichia coli. Infect. Immun. 55:2052-2056.
7. Camguilhem, R., and A. Milon. 1989. Biotypes and O serogroups of Escherichia coli involved in intestinal infections of weaned rabbits: clues to diagnosis of pathogenic strains. J. Clin. Microbiol. 27:743-747.
8. Iguchi, A., T. Ooka, Y. Ogura, Asadulghani, K. Nakayama, G. Frankel, and T. Hayashi. 2008. Genomic comparison of the O-antigen biosynthesis gene clusters of Escherichia coli O55 strains belonging to three distinct lineages. Microbiology 154:559-570.
9. Simpson, N., R. Shaw, V. F. Crepin, R. Mundy, A. J. FitzGerald, N. Cummings, A. Straatman-Iwanowska, I. Connerton, S. Knutton, and G. Frankel. 2006. The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoea. Mol. Microbiol. 60:349-363.
10. Berger, C. N., V. F. Crepin, M. A. Jepson, A. Arbeloa, and G. Frankel. 2009. The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics. Cell. Microbiol. 11:309-322.
11. Kenny, B. 2001. The enterohaemorrhagic Escherichia coli (serotype O157:H7) Tir molecule is not functionally interchangeable for its enteropathogenic E. coli (serotype O127:H6) homologue. Cell. Microbiol. 3:499-510.
12. Donnenberg, M. S., J. Yu, and J. B. Kaper. 1993. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol 175:4670-4680.
13. Lai, L. C., L. A. Wainwright, K. D. Stone, and M. S. Donnenberg. 1997. A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells. Infect Immun 65:2211-2217.
14. Kenny, B., L. C. Lai, B. B. Finlay, and M. S. Donnenberg. 1996. EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells. Mol Microbiol 20:313-323.
15. Donnenberg, M. S., and J. B. Kaper. 1991. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infection and immunity 59:4310-4317.
16. Schlosser-Silverman, E., M. Elgrably-Weiss, I. Rosenshine, R. Kohen, and S. Altuvia. 2000. Characterization of Escherichia coli DNA lesions generated within J774 macrophages. J. Bacteriol. 182:5225-5230.
17. Munera, D., V. F. Crepin, O. Marches, and G. Frankel. 2010. N-terminal type III secretion signal of enteropathogenic Escherichia coli translocator proteins. J. Bacteriol. 192:3534-3539.
18. Nougayrede, J. P., and M. S. Donnenberg. 2004. Enteropathogenic Escherichia coli EspF is targeted to mitochondria and is required to initiate the mitochondrial death pathway. Cell Microbiol 6:1097-1111.
19. Crepin, V. F., R. Shaw, C. M. Abe, S. Knutton, and G. Frankel. 2005. Polarity of enteropathogenic Escherichia coli EspA filament assembly and protein secretion. J Bacteriol 187:2881-2889.
20. Clements, A., K. Smollett, S. F. Lee, E. L. Hartland, M. Lowe, and G. Frankel. 2011. EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function. Cell Microbiol 13:1429-1439.
21. Mallick, E. M., M. J. Brady, S. A. Luperchio, V. K. Vanguri, L. Magoun, H. Liu, B. J. Sheppard, J. Mukherjee, A. Donohue-Rolfe, S. Tzipori, J. M. Leong, and D. B. Schauer. 2012. Allele- and tir-independent functions of intimin in diverse animal infection models. Front Microbiol 3:11.
![· Web viewPCL is a FDA-approved polymer [19] for hard and soft tissue applications, biocompatible and miscible with several polymers, easily processed and moulded, properties that](https://static.fdocument.org/doc/165x107/5e2e4d5008ee0663e865ba53/web-view-pcl-is-a-fda-approved-polymer-19-for-hard-and-soft-tissue-applications.jpg)
