Time course of accumulation of AIS proteins during neuron development. (A–D) Major AIS proteins remain associated with the cytoskeleton after detergent extraction of DIV14 neurons and colocalize with AnkG. (E) Adducin is largely absent from the AnkG-containing AIS (arrowhead), but is abundant in dendrites and in the axon outside of the AIS (arrow). (F) Time course of protein accumulation in the AIS during neuronal development. Fluorescence intensities of individual immunostained proteins are expressed as a percentage of the maximal value over a period from DIV4 to DIV21 (DIV7 for adducin and DIV21 for other proteins) and plotted against the DIV. n values for DIV4, DIV6/7, DIV10, DIV14, and DIV21 are, respectively: 21, 29, 38, 36, and 51 for spectrin βIV; 9, 18, 28, 26, and 34 for NrCAM; 22, 37, 24, 46, and 32 for neurofascin; 36, 29, 27, 28, and 64 for Nav; and 98, 123, 127, 146, and 190 for AnkG. For adducin, n = 10 for DIV4–14 and 9 for DIV21. Error bars represent standard deviations. Bars: (A–D) 20 µm; (E) 10 µm.