Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin

Abstract

<jats:title>Abstract</jats:title><jats:p>The lack of tools to reliably detect RanBP9 <jats:italic>in vivo</jats:italic> 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.</jats:p><jats:p>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 αtag 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 <jats:italic>in vivo</jats:italic> such as those linked to Nucleolin.</jats:p>

Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex‐dependent regulation of glycolysis

“…Size of the node reflects size of the term, and color reflects q-value ranging from orange (0.05) to white (0) identified, including 9 previously reported to associate with subunits of the CTLH complex and 76 novel interactors (Figure 3A,B, Supplemental File S4). 17,23,[36][37][38][39] Among those CTLH-associated proteins were the coREST transcriptional repressor complex, ribosomal proteins, heat shock chaperones, lysosomal proteins, RNA binding proteins, proteins associated with desmosomes, and glycolysis enzymes (Figure 3A). GO cellular compartment analysis revealed that the associated proteins were near equally associated with the cytoplasm and nucleus, consistent with the complex being present in both compartments, 15,16 with enrichment as well in extracellular organelles, focal adhesion, and vesicles (Figure 3C).…”

RANBP9 as potential therapeutic target in non-small cell lung cancer

“…This model has been validated through the use of immunohistochemistry and by coimmunoprecipitation, showing that the expression and interactions of the tagged protein faithfully recapitulates those of the wild type RANBP9. This new murine strain will be instrumental in obtaining data in vivo about the RANBP9immunocoprecipated proteome, without the risk of using antibodies that may recognize RANBP10 [84] .…”