Figure 5

Microtubule depolymerization inhibits netrin-1 induced phosphorylation of EF2K and EF2. Cultured stage 24/25 retinal explants were treated with the mTOR inhibitor rapamycin or a vehicle only control (A, B). Rapamycin induced a significant reduction in the level of phosphorylation of a downstream target of mTOR, eEF2 kinase (A-C). Quantitative immunofluorescence was also used to assess the levels of phosphorylation of both eEF2 kinase (D) and eEF2 (E) in response to netrin-1 stimulation. Netrin-1 induced a significant increase in the fluorescence intensity of both phospho-eEF2 kinase (D) and phospho-eEF2 (E) in growth cones treated with a DMSO vehicle control (ctrl). However, no significant changes in fluorescence intensity following netrin-1 stimulation were observed following inhibition of the microtubule cytoskeleton with colchicine (colc; (D, E)). The number of growth cones analyzed in each treatment group can be found in the corresponding bar of the respective graphs. ns = not significant. ***P < 0.0005 Mann-Whitney test. Scale bar 10 μm.