Ci-VSP forms dimers. (a) Schematic of the SiMPull subunit counting assay depicting the HA-VSP-GFP expression in HEK293 cells followed by pull down of the HA tag with anti-HA on coverslips and recording of the GFP fluorescence using TIRF. TIR, total internal reflection. (b) Left: TIRF images of HA-VSP-GFP single molecules. Middle: Representative trace showing two-step photobleaching of HA-VSP-GFP. AU, arbitrary units. Right: Summary of photobleaching step distribution for HA-VSP-GFP. Two-step bleaching events indicate VSP forms dimers on the membrane. (c) SiMPull data for trimer control, HA-GFP-ASIC1A. Left: TIRF images. Middle: Representative trace showing three-step photobleaching. Right: Summary of photobleaching step distribution. (d) SiMPull data for dimer control, HA-GFP-TREK1. Left: TIRF images. Middle: Representative trace showing two-step photobleaching. Right: Summary of photobleaching step distribution. (e) SiMPull data for monomer control, HA-TREK1 pull down of TREK1-GFP. Left: TIRF images. Middle: Representative trace showing single-step photobleaching. Right: Summary of photobleaching step distribution. Error bars are ±SEM. Bars, 2 μm.