Fig. 2

Effects of chymotrypsin on protein expression. Protein expression in extracts of brain slices is summarised as image densities (arbitrary units) of Western blots quantified using Image J for the effects of chymotrypsin on (a) neogenin, (b) DCC, (c) Unc-5C, (d) Unc-5A, (e) Shh and (f) RhoA expression. Sample blots are shown below each chart, which illustrate the concentration-dependent effects of chymotrypsin. Chart (g) shows the depletion of DCC by the chymotryptic protein cathepsin G in tissue slices. Panels (h) illustrate the agarose spot assay to study the migration of SH-SY5Y cells towards netrin-4. The spot border is indicated by the dotted line. The accumulation of cells on the outside of a normal spot is shown in the top micrograph (control), with the penetration of cells into the spot induced by netrin-4 (netrin, middle micrograph) and reduced penetration in the presence of chymotrypsin (netrin + CT 100nM). The number of cells entering the spots is summarised in chart (i). Photograph (j) is of SH-SY5Y cells used for the measurement of neurite growth after staining with cresyl violet, with bipolar and multipolar cells visible. The accompanying bar charts summarise the measurements of soma diameter (k), neurites per cell (l), mean neurite length (m) and the proportion of neurites with primary branching (n). Bars in charts (a) to (i) represent mean ± s.e.mean (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 relative to the control bar. Calibration bars: 200 μm in (h), 25 μm in (j)