Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1
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AbstractIntramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical in vitro biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation assay (PLA). PLA is a specific and sensitive method that allows the observation of interacting proteins by target-specific antibody detection coupled to rolling circle amplification. ELMO1 is the regulatory subunit of ELMO1-dedicator of cytokinesis 180 (DOCK180) bipartite Rac1 guanine nucleotide exchange factor (GEF) which adopts a closed autoinhibitory conformation via an intramolecular interaction of its N-terminal ELMO inhibitory domain (EID) and C-terminal ELMO autoregulatory domain (EAD). In the assay, PLA signals are detected in cells transfected with ELMO11-315 and ELMO1315-727 fragments. Moreover, overexpression of FE65, a neuronal adaptor which has been shown to disrupt ELMO1 intramolecular interaction, reduces the PLA signals of the two ELMO1 fragments significantly. Together, our results demonstrate that PLA can be employed for studying protein intramolecular interaction.
All Author(s) ListWai Wa Ray Chan, Dik Long Dennis Chau, Wen Li, Kwok-Fai Lau
Journal nameBio-protocol
Volume Number9
Issue Number23
Article numbere3449
LanguagesEnglish-United States
KeywordsAutoinhibition, Engulfment and cell motility 1, Fluorescence, Intramolecular interaction, PLA

Last updated on 2020-30-06 at 02:05