This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. EGFR (the epidermal growth factor receptor) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands, being activated by binding to epidermal growth factor and transforming growth factor ? (TGF?). Mutations that lead to EGFR overexpression have been associated with a number of cancers, including lung cancer and glioblastoma multiforme. To isolate EGFR with its interacting partners we tested several monoclonal and polyclonal commercially available anti-EGFR antibodies. However, none of these proved successful for immunoisolations. We therefore proceeded in testing in-house-developed antibodies (rabbit polyclonal), which we had to further purify to achieve antibodies usable for immunoisolations. After careful optimization of lysis buffers, and tests of magnetic beads versus agarose beads, we now have isolated EGFR with associated proteins. Our results confirmed previous known interacting partners, such as GRB2, and indicated numerous phosphorylation sites on EGFR. In our effort to obtain immunoisolations as clear as possible of background (non-specific binding), we tested the use of a filtration step prior to immunoisolations. Following filtration, we could still detect EGFR and GRB2. Having successfully isolated EGFR, we have now started the comparison between wild type and two mutant EGFR proteins.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000862-38
Application #
8361534
Study Section
Special Emphasis Panel (ZRG1-BCMB-Q (40))
Project Start
2011-03-01
Project End
2012-03-31
Budget Start
2011-03-01
Budget End
2012-03-31
Support Year
38
Fiscal Year
2011
Total Cost
$1,304
Indirect Cost
Name
Rockefeller University
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C et al. (2017) Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding. Anal Biochem 519:38-41
Chait, Brian T; Cadene, Martine; Olinares, Paul Dominic et al. (2016) Revealing Higher Order Protein Structure Using Mass Spectrometry. J Am Soc Mass Spectrom 27:952-65
Boice, Michael; Salloum, Darin; Mourcin, Frederic et al. (2016) Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells. Cell 167:405-418.e13
Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Maximizing ion transmission from atmospheric pressure into the vacuum of mass spectrometers with a novel electrospray interface. J Am Soc Mass Spectrom 26:649-58
Mast, Fred D; Rachubinski, Richard A; Aitchison, John D (2015) Signaling dynamics and peroxisomes. Curr Opin Cell Biol 35:131-6
Krutchinsky, Andrew N; Padovan, Júlio C; Cohen, Herbert et al. (2015) Optimizing electrospray interfaces using slowly diverging conical duct (ConDuct) electrodes. J Am Soc Mass Spectrom 26:659-67
Oricchio, Elisa; Papapetrou, Eirini P; Lafaille, Fabien et al. (2014) A cell engineering strategy to enhance the safety of stem cell therapies. Cell Rep 8:1677-1685
Zhong, Yu; Morris, Deanna H; Jin, Lin et al. (2014) Nrbf2 protein suppresses autophagy by modulating Atg14L protein-containing Beclin 1-Vps34 complex architecture and reducing intracellular phosphatidylinositol-3 phosphate levels. J Biol Chem 289:26021-37
Xue, John Z; Woo, Eileen M; Postow, Lisa et al. (2013) Chromatin-bound Xenopus Dppa2 shapes the nucleus by locally inhibiting microtubule assembly. Dev Cell 27:47-59
Indiani, Chiara; O'Donnell, Mike (2013) A proposal: Source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 18:312-23

Showing the most recent 10 out of 67 publications