TY - JOUR
T1 - Evaluation of the selectivity and sensitivity of isoform-And mutation-specific RAS antibodies
AU - Waters, Andrew M.
AU - Ozkan-Dagliyan, Irem
AU - Vaseva, Angelina V.
AU - Fer, Nicole
AU - Strathern, Leslie A.
AU - Hobbs, G. Aaron
AU - Tessier-Cloutier, Basile
AU - Gillette, William K.
AU - Bagni, Rachel
AU - Whiteley, Gordon R.
AU - Hartley, James L.
AU - Mccormick, Frank
AU - Cox, Adrienne D.
AU - Houghton, Peter J.
AU - Huntsman, David G.
AU - Philips, Mark R.
AU - Der, Channing J.
N1 - Publisher Copyright:
Copyright © 2017 The Authors, some rights reserved.
PY - 2017/9/26
Y1 - 2017/9/26
N2 - 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 BRAFV600E. 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.
AB - 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 BRAFV600E. 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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U2 - 10.1126/scisignal.aao3332
DO - 10.1126/scisignal.aao3332
M3 - Article
C2 - 28951536
AN - SCOPUS:85030178665
VL - 10
JO - Science's STKE : signal transduction knowledge environment
JF - Science's STKE : signal transduction knowledge environment
SN - 1937-9145
IS - 498
M1 - eaao3332
ER -