The Journal of Neuroscience, January 12, 2005, ():
Axonal Protein Synthesis and Degradation Are Necessary for Efficient Growth Cone Regeneration
J. Neurosci. Verma et al.
25: 331
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Fig. S1. Specificity of the anti-ribosomal-P0,-phospho eIF 4E, -20s proteasome, -ubiquitin, and –ubiquitinated protein (FK2) antibodies was evaluated by immunoblotting. Each antibody preparation recognized major bands of the expected molecular weight. Similar data have been published for the anti-ubiquitin, and – ubiquitinated protein (FK2) antibodies (Fujimuro et al., 1994).
- supplemental material
-
Fig. S2 (a). Quantification of relative intensities of immunofluorescence. Axons were selected at random and the intensity of the pixels contained within the demarcated area measured for the antibody of interest. Intensities from a negative control and the intensity of background staining were also measured and these values removed from the axonal staining intensity. This value was then ratioed to the intensity of tubulin staining for the identical region on the axon to give the final intensity reading. (b) Ratioing of intensities relative to tubulin reduced the variance of the data, but did not change the overall differences measured for the various molecules between different axon types. The two histograms show data for ribosomal P0 levels plotted with and without ratioing to tubulin. Relative levels of immunostaining obtained for all the antigens were similar to those where the intensities were not corrected for axon dimensions with tubulin. (c) Intensity of ?-tubulin immunoreactivity does not vary with axonal type or distance from the growth cone.
- supplemental material
-
Fig. S3. Percentage reduction in growth cone regeneration of cell-body attached and axon-only sensory and retinal cultures compared to control (no inhibitor) exposed to cycloheximide, anisomycin, lactacystin, LnLL, rapamycin, SB203580, C3I, C3 I2 and C2 I9. For all experiments, the effects of the compounds were tested on axons that were still attached to their cell bodies, axotomised 100µm or more away. In order to demonstrate that the effects were directly on the axons rather than on the cell bodies, all the experiments were repeated on adult DRG axons whose cell bodies had been removed just before the experiment. The effects of all the treatments was similar for axon-only and cell-body attached adult DRG cultures.
