Fig. 8. Internalisation of L1 and NRP1 is affected in TAG-1 mutants. Immunolabelling of growth cones from wild-type (A,C,E,G; WT) and TAG-1-null (B,D,F,H; null) sensory neurons shows that L1 (A,B) and NRP1 (C,D) reach the surface of TAG-1-null growth cones at normal levels, as quantitated in I (L1) and J (NRP1); n=16 for wild type, n=21 for null (see Materials and methods). Quantitation of surface immunolabelling after Sema3A treatment of wild-type and TAG-1-null growth cones shows that surface levels of L1 (E,F,I) and NRP1 (G,H,J) are significantly reduced in wild type (E,G; WT + Sema) but not TAG-1-null (F,H; Null + Sema) growth cones (^**P<0.01, ^*P<0.05; unpaired t-test). Results from both collapsed and extended growth cones are pooled for each genotype in both conditions (and are present in each population in the proportions indicated in Fig. 4I) but results are expressed as average intensity per unit area to normalise for differences in growth cone size [similar results were obtained when comparing growth cones from the different genotypes according to morphology (i.e. extended versus collapsed; not shown)]. (K) FITC-labelled dextran is taken up to a lesser extent by TAG-1-null growth cones (null + Sema) than by wild-type growth cones (WT + Sema) after Sema3A treatment. (L) Cell-surface TAG-1 levels (red) in wild-type growth cones (counterlabelled with phalloidin; green) fall significantly (P<0.0001) after Sema3A treatment (+Sema; n=13) compared with no treatment (NT; n=12). Panels on the right in each pair show TAG-1 only. Quantitation as for L1 and NRP1 above. (M-Q) Colocalisation of TAG-1 and clathrin heavy chain in wild-type growth cones after Sema3A treatment. Area outlined by white box in P shown in Q. Scale bars: 10 µm in A-H,Q; 20 µm in L.