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Biology

La base di Cell-L-Glutatione Protezione saggi per studiare endocitosi e riciclo dei Plasma proteine ​​di membrana

Published: December 13, 2013 doi: 10.3791/50867

Materials

Name Company Catalog Number Comments
CFBE41o- cells reference10,11,13,14,15
HEK293 cells reference13
Minimal Essential Medium (MEM) Gibco 11095
L-Glutamine Cellgro 25-005-CL
Penicillin Sigma P0781
Streptomycin
Puromycin InvivoGen ant-pr-1
PureCol Bovine Collagen Solution Type I Advanced Biomatrix 5005-B
Culture Reagents
Dulbecco’s Phosphate Buffered Saline (PBS) Gibco 14190
Calcium chloride Sigma C3306
Magnesium chloride Sigma M2670
Sodium chloride Sigma S3014
EZLink Sulfo-NHS-SS-Biotin Pierce 21331
L-Glutathione reduced (GSH) Sigma G6529
Streptavidin-agarose resin Thermo Fisher 20353
Sodium hydroxide Sigma 224165
Fetal bovine serum  Gemini Bioscience 100-106
Milli-Q water Millipore Advantage System
HEPES Sigma H3375
Triton X-100 Bio Rad 161-0407
Glycerol FisherScientific G33
Laemli Sample buffer BioRad 161-0737
Dithiothreitol (DTT) Sigma 646563
Complete Protease Inhibitor cocktail Roche 1697498
Streptavidin beads Thermo Scientific 20353
Chemiluminescence Perkin Elmer NEL104001EA
Reagents
Nonfat dry milk Carnation N/A
Tween-20 BioRad 170-6539
Tris-buffered Saline (TBS) BioRad 170-6435
Bovine Serum Albumin (BSA) Gibco 15120
Primary and secondary 
Antibodies for Western Blotting
Mouse monoclonal Anti-CFTR 596 Cystic Fibrosis Foundation Therapeutics, Inc., Chapel Hill, NC
Anti-ezrin 
Running buffer 10x BioRad 161-0772
Transfer buffer 10x (without SDS) BioRad 161-0771
Methanol Fisher A4129
Coulter counter Beckman Coulter Z1Coulter Particle Counter
Standard Cell Culture equipment
Cell culture hood with UV light
Stock flasks
Transwell filters-24mm Corning 3412
4 cm cell culture dishes
pH meter
15 ml and 50 ml conical tubes
1.5 ml Eppendorf tubes
Rubber policeman
Cold room
Ice buckets and ice
27 G needles
37 ºC Incubator
Thermometer
Benchtop centrifuge
Cooled centrifuge
Electrophoresis equipment
Developer
Film GE Amersham 28-9068-36
PVDF membrane Millipore IPVH00010
ImageJ software NIH

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References

  1. Sigismund, S., et al. Endocytosis and signaling: cell logistics shape the eukaryotic cell plan. Physiol. Rev. 92, 273 (2012).
  2. Andersson, E. R. The role of endocytosis in activating and regulating signal transduction. Cell Mol. Life Sci. 69, 1755 (2012).
  3. Grant, B. D., Donaldson, J. G. Pathways and mechanisms of endocytic recycling. Nat. Rev. Mol. Cell Biol. 10, 597 (2009).
  4. Bretscher, M. S., Lutter, R. A new method for detecting endocytosed proteins. EMBO J. 7, 4087 (1988).
  5. Watts, C., Davidson, H. W. Endocytosis and recycling of specific antigen by human B cell lines. EMBO J. 7, 1937 (1988).
  6. Graeve, L., Drickamer, K., Rodriguez-Boulan, E. Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor. J. Cell Biol. 109, 2809 (1989).
  7. Garza, L. A., Birnbaum, M. J. Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes. J. Biol. Chem. 275, 2560 (2000).
  8. Le, T. L., Joseph, S. R., Yap, A. S., Stow, J. L. Protein kinase C regulates endocytosis and recycling of E-cadherin. Am. J. Physiol. Cell Physiol. 283, C489 (2002).
  9. Swiatecka-Urban, A., et al. PDZ-domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 277, 40099 (2002).
  10. Swiatecka-Urban, A., et al. Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 279, 38025 (2004).
  11. Swiatecka-Urban, A., et al. The short apical membrane half-life of rescued uF508-CFTR results from accelerated endocytosis uF508-CFTR in polarized human airway epithelial cells. J. Biol. Chem. 280, 36762 (2005).
  12. Swiatecka-Urban, A., et al. Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells. Am. J. Physiol. Cell. Physiol. 290, C862 (2006).
  13. Swiatecka-Urban, A., et al. Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells. J. Biol. Chem. 282, 23725 (2007).
  14. Ye, S., et al. c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells. J. Biol. Chem. 285, 27008 (2010).
  15. Cihil, K. M., et al. Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells. J. Biol. Chem. 287, 15087 (2012).
  16. MacEachran, D. P., et al. The Pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator. Infect. Immun. 75, 3902 (2007).
  17. Pagano, A., Crottet, P., Prescianotto-Baschong, C., Spiess, M. In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5. Mol. Biol. Cell. 15, 4990 (2004).
La base di Cell-L-Glutatione Protezione saggi per studiare endocitosi e riciclo dei Plasma proteine ​​di membrana
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Cite this Article

Cihil, K. M., Swiatecka-Urban, A.More

Cihil, K. M., Swiatecka-Urban, A. The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins. J. Vis. Exp. (82), e50867, doi:10.3791/50867 (2013).

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