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A proteomic approach for dissecting H‐Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells
Author(s) -
Park Jung Wook,
Kim Seyoon,
Bahk Young Yil
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500688
Subject(s) - proteome , biology , 3t3 cells , oncogene , microbiology and biotechnology , embryonic stem cell , proteomics , western blot , effector , fibroblast , signal transduction , cell culture , cell , cell cycle , transfection , genetics , gene
To elucidate an understanding into H‐Ras protein network, we have established various oncogene H‐Ras‐expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H‐Ras, G12R H‐Ras, and G12V/T35S H‐Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function H‐Ras‐expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2‐DE, quantitative image analysis and MALDI‐TOF‐MS analysis both commonly in oncogenic and partial loss of function H‐Ras expression system. Thus, we tried to dissect H‐Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H‐Ras and a partial loss of function H‐Ras in the H‐Ras‐expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2‐DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT‐PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H‐Ras‐expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.