Figure 1.

Amphoterin protein is enriched in axons of injury-conditioned neurons. A, Axonal versus cell body compartments were used for isolation of protein from DRG cultures. By immunoblotting, amphoterin protein is shown to be cell body predominant in naive DRG cultures and axon predominant in injury-conditioned DRG cultures. Replicate blots probed with anti-Erk 1/2 shows approximately equal loading between the naive and injury-conditioned lysates. B, C, Representative, exposure-matched epifluorescent images of naive and injury-conditioned DRG cultures stained for amphoterin (red) and NF (green) protein are shown in B. Consistent with the immunoblotting in A, amphoterin protein is higher in cell bodies of naive compared with injury-conditioned neurons. There is a clear increase in amphoterin protein in the axons of the injury-conditioned neurons and the signal appears concentrated along the periphery of the axon (arrowheads). Cell body signals are noted along the cell periphery (arrows) and in the nucleus of the naive neurons, whereas the injury-conditioned neurons show predominantly cell periphery signals (arrows). Quantification immunoreactivity from exposure-matched image sets in C shows increased levels of amphoterin protein in axons of injury-conditioned versus naive DRG cultures, whereas cell bodies show a decrease in amphoterin protein in injury-conditioned versus naive DRG cultures (n ≥ 25 for cell body and n ≥ 30 for axons from at least 3 separate experiments; **p ≤ 0.01, ***p ≤ 0.001 by Student's t test) Scale bars, 10 μm. D, Representative, exposure-matched confocal images of L4 DRGs and sciatic nerves of naive and 7 d postsciatic nerve crush animals with the amphoterin protein in red and the NF protein in green. Similar to the cultured neurons in B, there is a clear increase in axonal amphoterin protein (arrows) in the injured compared with naive nerve. Amphoterin protein signals in the neuronal perikaryon also decrease in the injured compared with naive DRGs. Satellite cells in the DRG show a strong, apparently nuclear signal for amphoterin that similarly declines with injury (arrowheads). E, Quantification of amphoterin protein signals from the tissue sections comparing axonal levels in naive versus injured nerve and neuronal cell body levels in naive versus injured DRGs (n ≥ 25 for cell body and n ≥ 30 for axons from at least 3 separate experiments; **p ≤ 0.01, ***p ≤ 0.001 by Student's t test). Scale bars: DRG panels, 25 μm; sciatic nerve panels, 10 μm.