Figure 6.

Effects of antisense oligonucleotide treatment on hnRNP A2 distribution and trafficking of A2RE-containing RNA. A, Western blot of hnRNP A2 in sense and antisense oligonucleotide-treated neurons with α-tubulin used as control for protein loading on the gel. B, Intracellular distribution of hnRNP A2 in hippocampal neurons after hnRNP A2 sense and antisense treatment. In sense-treated cells, the protein is observed in the soma (arrowhead) and as granules in the processes (arrows). In antisense-treated cells, the hnRNP A2 is confined primarily to the nucleus (arrowheads), with markedly lower levels in the processes (arrows). The same confocal microscopy data collection and image processing parameters were used for both samples. In both images the maximal brightness in the nucleus has been set just below 255, ensuring that all pixels are within the dynamic range of the imaging system, permitting direct comparison of the relative fluorescence intensities in the neurites in the two images. C, Representative images of hippocampal neurons microinjected with fluorescently labeled RNAs containing A2RE or the A2RE-like element of MAP2A after hnRNP A2 sense and antisense treatment. Injected RNAs were detected 50 μm or more from the cell bodies in sense-treated cells (right panels, arrows). In antisense-treated cells, RNAs were observed only in the proximal processes (left panels, arrowheads). D, Percentage of transport-positive, sense- and antisense-treated cells injected with RNA containing A2RE or the cognate sequence from MAP2A RNA. Error bars represent the SDs. Scale bar, 15 μm.