Figure 4. Overexpression of ankyrins and spectrins shifts the intra-axonal barrier.

(A) Cultured hippocampal neuron transfected with ankB-GFP at DIV 0 and immunostained for GFP, ankG, and MAP2 at DIV 7. Scale bar, 20 μm.

(B) Quantification of AIS length (ankG immunoreactivity) after transfection of the indicated proteins.

(C–D) Cultured hippocampal neuron transfected with αII spectrin-GFP at DIV 0 and immunostained for GFP, ankG (C) or ankB (D), and MAP2 at DIV 7. Brackets indicate the proximal axon, and arrowheads indicate the growth cone. Scale bar, 20 μm.

(C) (E–F) Cultured hippocampal neuron transfected with βII spectrin-GFP at DIV 0 and immunostained for GFP, ankG (C) or ankB (D), and MAP2 at DIV 7. Brackets indicate the proximal axon, and arrowheads indicate the growth cone. Scale bar, 20 μm.

(G–H) Cultured hippocampal neurons transfected with ankG270-GFP at DIV 0, immunolabeled for GFP and ankB at DIV 3. Arrowhead in (G) indicates the end of the axon. Scale bar, 25 μm.

(I-J) Cultured hippocampal neurons transfected with ankG270-GFP at DIV 0, immunolabeled for GFP and ankB (at DIV 7). Arrowheads indicate the location of the intra-axonal boundary. Scale bar, 50 μm.

(K) Immunoblots of DIV 7 cultured hippocampal neurons infected with adenovirus to silence expression of the indicated proteins. Each lane corresponds to proteins from two replicate wells. Neurofilament-M (NFM) was used as a loading control.

(L–N) Hippocampal neurons infected at DIV 0 with adenovirus to silence expression of ankB (L), αII spectrin (M), and βII spectrin (N), and labeled for GFP (green), MAP2 (blue), and Tau1 (red) at DIV 7. Scale bars, 25 μm.

(O–P) Hippocampal neurons infected at DIV 0 with adenovirus to silence expression of αII spectrin (O) or βII spectrin (P), and labeled for ankB, GFP, and MAP2 at DIV 7. Scale bars, 25 μm.