Visualization of membrane insertion events. (A) TIRFM image of a Neuro-2a cell transfected with α6-SEPβ2β3 nAChRs and excited with a 488-nm laser. Images were taken with a frame rate of 200 ms. (B₁) Frame directly preceding an insertion event at the PM. (B₂) Frame showing the insertion and (B₃) the frame directly after the observation of the insertion event. The white circle marks the location of the insertion. (C and D) Frequency of insertion events for α6-SEPβ2* nAChRs without or with nicotine treatment (50 nM, 24 h) calculated as insertion events per minute (n = 4). Bars, 10 µm. Data are mean values ± SEM. n.s., not significant; *, P < 0.05.