Fig. 3. S1P-induced responses are sensitive to inhibitors of proteasomal degradation. (A) S1P-induced growth cone collapse (stage 32 embryos cultured for 24 hours) is blocked by proteasome inhibitors (LnLL, Lacta). Conversely, translation inhibitors (CHX, Anisomycin) had no effect on S1P-induced growth cone collapse. ^*P<0.05, Mann-Whitney U test. (B,C) Cumulative distribution of turning angles after LnLL and Lacta treatment. S1P-induced repulsive turning of growth cones is blocked by proteasomal inhibitors. (C) Mean turning graph from data in C. ^***P<0.005, Kolmogorov-Smirnov test. (D,E) Quantitative immunofluorescence analysis using antibodies directed against ubiquitin-protein conjugates (FK2) or the phosphorylated form of eIF4EBP-1 (eIF4EBP-1-P). (D) Representative pictures of FK2 and eIF4EBP-1-P immunoreactivities within growth cones. (E) S1P caused a significant increase in FK2 signal when compared with the control level, whereas the eIF4EBP-1-P signal intensity remained unchanged. Numbers inside bars indicate growth cones tested. ^***P<0.005, Mann-Whitney U test. n.s., non significant. Scale bar in D: 10 µm.