KD and replacement of endogenous Sec8 enables rSec8-TagRFP to be incorporated into the exocyst and visualization of small dynamic puncta by TIRFM. (A) HeLa cells were transfected with rSec8TagRFP and either scrambled (Scram) RNAi or RNAi to Sec8 for 60 h and immunoblotted against Sec8, which showed that rSec8-TagRFP was stabilized after Sec8KD. (B) IP of TagRFP pulled down other exocyst subunits in “Sec8-replaced” stable cells (Sec8KD plus rSec8TagRFP). (C) Live-cell TIRFM showed that rSec8-TagRFP localized to small dot-like structures in Sec8-replaced cells. (left) Cells transfected with Scram RNAi and rSec8-TagRFP showed aggregates (arrows) or cytoplasmic localization (asterisk). (right) KD rSec8-TagRFP in Sec8-replaced cells showed vesicle-like structures (arrowheads; maximum projection of Video 1). (D) Kymograph of box in C. rSec8-TagRFP puncta appeared (open arrowheads), remained static, and disappeared (closed arrowheads), indicative of potential tethering.