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. 2012 Apr 27;7(4):e36023. doi: 10.1371/journal.pone.0036023

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A. ITSN1 interacts with Arf6 as well as its effector Arfaptin2. BiFC was used to analyze the interaction of ITSN1S with components of the Arf6 pathway. VN-ITSN1-S was co-expressed in COS cells with VC-Arf6 (top panels), VC-Arfaptin2 (middle panels), or VC-pep (bottom panels) as a negative control. The emission from the reconstituted Venus fluorophore was pseudocolored green. CFP (pseudocolored red) was co-transfected to mark transfected cells and only CFP-positive cells were imaged. Scale bar, 20 um. The fluorescence pattern shown in all panels are representative of localization observed throughout the plate. B. ITSN1 forms a trimolecular complex with Arf6 and Arfaptin2 in COS cells. Top panels: VN-ITSN1-S and VC-Arf6 were co-expressed in the absence of CFP-Arfaptin2 to demonstrate lack of bleed through of the BiFC signal into the CFP channel. Middle panels: CFP-Arfaptin2 was expressed alone to demonstrate lack of bleed through of the CFP signal into the BiFC channel. Bottom panels: VN-ITSN1-S, VC-Arf6, and CFP-Arfaptin2 were co-expressed in COS cells. The BiFC complex of VN-ITSN1-S and VC-Arf6 co-localizes with CFP-Arfaptin2. A region of overlap of the BiFC complex with CFP-Arf6 is enlarged in the inset in the bottom right panel. Note: white scale bar represents 20? m. The fluorescence pattern shown in all panels are representative of localization observed throughout the plate. C. VC-Arfaptin2 co-precipitates with ITSN1. HA-epitope tagged ITSN1-S or various domains of ITSN1 were co-expressed with VN-Arfaptin2 which contains a FLAG epitope tag. HA-immunoprecipitates were probed for either HA (top panel) or FLAG (bottom) to detect association of Arfaptin2. The CC region interacts most strongly with VN-Arfaptin2 consistent with the Y2H results (see Tables S1).