Journal
SCIENCE SIGNALING
Volume 10, Issue 498, Pages -Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/scisignal.aao3332
Keywords
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Categories
Funding
- NIH/NCI [T32 CA009156, F32 CA200313]
- Pancreatic Cancer Action Network-American Association for Cancer Research
- Department of Defense
- Lustgarten Pancreatic Cancer Foundation
- NIC/NIH [HHSN261200800001E]
- [CA42978]
- [CA179193]
- [CA175747]
- [CA199235]
- [CA197709]
- [CA165995]
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There is intense interest in developing therapeutic strategies for RAS proteins, themost frequentlymutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan-or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from Rasless cells expressing the oncoprotein BRAF(V600E). Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.
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