Figure 5. Antisense Oligonucleotides to the ß-Actin Zipcode Inhibit the Localization of ZBP1

Cultured neurons were starved in MEM for 4 hr and then stimulated with NT-3 for 10 or 60 min in the presence of either antisense or control oligonucleotides. ZBP1 (green, detected by antibodies) was prominent in the cell body but not appreciable in distal neurites or growth cones after the starvation assay (not shown). Addition of NT-3 to the starvation medium for (A) 10 or (B) 60 min, in the presence of the antisense oligonucleotides, failed to show ZBP1 localization in distal segments and growth cones, although granules were observed within the proximal segment (arrows). In the presence of the control oligonucleotides, addition of NT-3 for (C) 10 or (D) 60 min demonstrated strong induction of ZBP1 localization to distal neurites and growth cones (arrows).

(E) Quantitation of cells in (A)–(D) showed significantly higher fluorescence (pixel) intensities for ZBP1 in (C) and (D) than (A) and (B). (*, p < 0.05 for 10 min treatment; p < 0.01 for 1 hr treatment).

(F) Cells transfected with EGFP-ZBP1 showed an increase in fluorescence intensities in proximal portion of the neurites with increased time in NT-3, in the presence of control oligonucleotides. As in (E), a reduction in ZBP1 fluorescence intensities was observed in the neurite over time when treated with antisense.