Measurement of rab35 activity with the gtp-rab35 trapper rbd350

Hotaka Kobayashi, Kan Etoh, Soujiro Marubashi, Norihiko Ohbayashi, Mitsunori Fukuda

    Research output: Contribution to journalArticlepeer-review

    8 Citations (Scopus)

    Abstract

    Small GTPase Rab35 functions as a molecular switch for membrane traffi cking, specifi cally for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane traffi cking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (R ab- b inding d omain specifi c for Rab 35). Because RBD35 specifi cally interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identifi ed in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.

    Original languageEnglish
    Pages (from-to)207-216
    Number of pages10
    JournalMethods in Molecular Biology
    Volume1298
    DOIs
    Publication statusPublished - 2015

    Keywords

    • Neurite outgrowth
    • RBD35
    • RUN domain
    • RUSC2
    • Rab35

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

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