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. 2018 May 7;150(5):683–696. doi: 10.1085/jgp.201812064

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© 2018 Rayaprolu et al.

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Figure 4. — Ci-VSP subunits pull each other down consistent with a dimer complex. (a) Schematic of the SiMPull assay where HA-VSP is coexpressed with VSP-GFP in HEK293 cells followed by pull down of the HA-VSP with anti-HA on coverslips. Any HA-VSP dimers will not be visible, and VSP-GFP dimers do not interact and are washed away, leaving the HA-VSP with VSP-GFP dimer as the only visible fluorescent spot using TIRF. TIR, total internal reflection. (b) SiMPull TIRF images of the HA-VSP alone (left), VSP-GFP alone (middle), and HA-VSP/VSP-GFP mixture (right). Fluorescent spots are only visible in the HA-VSP/VSP-GFP sample. Bars, 2 μm. (c) Representative trace showing single-step photobleaching of the HA-VSP/VSP-GFP spots in b. AU, arbitrary units. (d) Summary of the photobleaching step distribution of the HA-VSP/VSP-GFP spots in b. As expected, monomers predominate the mixture, indicating that a HA-VSP subunit pulls down a VSP-GFP subunit, forming a dimer complex. Error bars are ±SEM.