Figure 2

Spatial localization of Nedd4-binding protein 3 in primary hippocampal neurons. Immunostaining of Nedd4-binding protein 3 (N4BP3) (Alexa Fluor 488 dye, green) in developing rat primary hippocampal neurons. (A) Colocalization of N4BP3 and the axonal growth cone (GC) marker protein 2G13P (Alexa Fluor 568 dye, red) in a stage 2 neuron. Note localization of N4BP3 at the GC base (framed arrowhead). (B) In stage 3 neurons at DIV3, N4BP3 is clearly localized within the axon, at axonal branching points and in axonal GCs. (C) N4BP3 (Alexa Fluor 488 dye, green; upper row) shows the same spatial distribution as interaction partner Nedd4 (Alexa Fluor 488 dye, green; lower row) within axonal GCs of stage 3 hippocampal neurons at DIV3. (D) and (E) N4BP3 labeling of both axonal branching points (D; framed arrowheads) and dendritic branching points (E; framed arrowheads) at DIV5 and DIV10, respectively. (F) Pie charts illustrating the percentage of branching points with (gray color) or without (white color) N4BP3 labeling in axons at DIV5 (left) and dendrites at DIV10 (right). (A) through (D) The actin cytoskeleton is visualized by phalloidin (Alexa Fluor 647 dye, magenta). (B), (D) and (E) Coimmunostaining with the dendritic marker protein microtubule-associated protein 2 (MAP2; Alexa Fluor 568 dye, red).