Na_v1.6-BAD-GFP is delivered along the length of the AIS where it is immediately immobilized.

<p><b>A)</b> Experimental outline. <b>B)</b> Representative TIRF image and reference for the Kymograph in (<b>E</b>). Dotted line indicates line scan starting in the soma (red) and continuing through the AIS (yellow). <b>C)</b> Representative images of SA-594 labeling of newly inserted channels in the AIS. Two SA-594 puncta appear in the axonal membrane previously devoid of surface labeling (yellow and red arrows), denoting insertion events. Two other puncta are present throughout this time course and show no lateral movement (blue arrows). <b>D)</b> Representative images of SA-594 labeling of newly inserted channels in the soma. The left panel shows the insertion site location (O) and subsequent single-molecule track overlaid on the region prior to the channel delivery at 4:11. <b>E)</b> Kymograph of the line scan shown in (<b>B</b>) indicating GFP (green) and SA-594 (red) fluorescence over time. <b>F)</b> Single-molecule tracking of inserted Na_v1.6-BAD-GFP. Each colored line represents an individual track. The black dotted line indicates the soma/AIS boundary. Neurons were transfected on DIV4 and imaged 36–44 hrs post-transfection. Scale bars represent 10 μm (B,E) or 2 μm (C,D,F).