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 |
References
- Sigismund, S., et al. Endocytosis and signaling: cell logistics shape the eukaryotic cell plan. Physiol. Rev. 92, 273 (2012).
- Andersson, E. R. The role of endocytosis in activating and regulating signal transduction. Cell Mol. Life Sci. 69, 1755 (2012).
- Grant, B. D., Donaldson, J. G. Pathways and mechanisms of endocytic recycling. Nat. Rev. Mol. Cell Biol. 10, 597 (2009).
- Bretscher, M. S., Lutter, R. A new method for detecting endocytosed proteins. EMBO J. 7, 4087 (1988).
- Watts, C., Davidson, H. W. Endocytosis and recycling of specific antigen by human B cell lines. EMBO J. 7, 1937 (1988).
- 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).
- Garza, L. A., Birnbaum, M. J. Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes. J. Biol. Chem. 275, 2560 (2000).
- 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).
- 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).
- Swiatecka-Urban, A., et al. Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator. J. Biol. Chem. 279, 38025 (2004).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).