Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo.

Soliman, S.; Stark, A. E.; Gardner, M.; Harshman, S. W.; Breece, C. C.; Amari, F.; Orlacchio, A.; Chen, M.; Tessari, A.; Martin, J. A.; Visone, R.; Freitas, M. A.; La Perle, K.; Palmieri, D.; COPPOLA, V.

doi:10.1101/745174

bioRxiv · 10.1101/745174

Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo.

Soliman, S.·Stark, A. E.·Gardner, M.·Harshman, S. W.·Breece, C. C.·Amari, F.·Orlacchio, A.·Chen, M.·Tessari, A.·Martin, J. A.·Visone, R.·Freitas, M. A.·La Perle, K.·Palmieri, D.·COPPOLA, V.

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Abstract

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a novel interaction with Nucleolin, a prominent nucleolar protein. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available tag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially reveal novel functions of this protein in vivo such as those linked to Nucleolin.

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Soliman, S., Stark, A. E., Gardner, M., Harshman, S. W., Breece, C. C., Amari, F., Orlacchio, A., Chen, M., Tessari, A., Martin, J. A., Visone, R., Freitas, M. A., La Perle, K., Palmieri, D., COPPOLA, V.. 2019-08-24. Tagging allows faithful tracing of expression and enhances biochemical detection of Ran Binding Protein 9 in vivo.. https://doi.org/10.1101/745174

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