Endogenous L1 mislocalizes to the somatodendritic domain when NEEP21 is down-regulated. (A and B) Dissociated neurons were electroporated with GFP as control (A and A′) or AS-NEEP21-GFP (B and B′) and surface L1 was detected with a polyclonal anti-L1 antibody (red) at DIV3. In GFP-expressing control cells (A and A′), endogenous L1 is highly polarized to the axon (arrows) and only weakly found on dendrites (arrowheads). In AS-NEEP21-GFP–expressing cells (B and B′), endogenous L1 is additionally detected on the soma and dendrites. (C) The A/D PI was determined for endogenous surface L1 for control cells expressing GFP or AS-NEEP21-GFP. n = 50 cells for GFP; n = 63 cells for AS-NEEP21-GFP from two independent experiments. Error bars indicate SEM. **, statistical significance at P < 0.0001. (D) DIV3 neurons were incubated with polyclonal anti-L1 antibody for 20 min and then fixed and stained with secondary antibody after permeabilization. Labeled endosomes can be seen in axons, somata, and dendrites. Two examples are shown.