A proteomic approach for unraveling the oncogenic H-Ras protein networks in NIH/3T3 mouse embryonic fibroblast cells.

To elucidate the oncogenic H-Ras network, we have established various stable and inducible oncogenic H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which express G12V H-Ras and G12R H-Ras proteins under the influence of a strong cytomegalovirus promoter and under the tight control of expression by an antibiotic, doxycycline, respectively. Here we provide a catalogue of proteome profiles in total cell lysates derived from oncogenic 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 using both a stable expression system as well as an inducible expression system. There were a large number of common targets for oncogenic H-Ras, which were identified in both cell lines and consisted of 64 proteins (36 up-regulated and 28 down-regulated). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, the results presented here show that comparative analysis of the proteome from the oncogenic H-Ras-expressing cells yielded interpretable data to elucidate protein networks directly and/or indirectly.