Figure 4
Molecular characterization of nGDF (TGF-β family member) in leech. (a) Nucleotide and amino acid sequences of leech nGDF. The numbers of nucleotides are indicated in left and of amino acids in right. The protein sequence of nGDF presents a propeptide and a mature form region, both framed. The nGDF protein sequence contains RRKR cut site and nine conserved cysteine residues, highlighted in dark grey and light grey, respectively. (b) Protein sequence showing the preserved pattern of TGF-β family in leech nGDF and sequence alignment with human GDF8 (O14793) and GDF11 (O95390). High and low consensus homologies are represented by red and blue residues, respectively. (c) Western blotting analysis from CNS protein extract using polyclonal rabbit anti-TGF-β antibodies (lane 1) compared to secondary antibody alone (lane 2) as a negative control (see also Supplementary Fig. S3 to have the overview of both membranes). (d) Immunofluorescence using polyclonal rabbit anti-TGF-β antibodies of nGDF in the point of lesion 15 minutes after-lesion, as framed in the CNS diagram. (e) No signal was detected in connective treated only with secondary antibody as negative control. Cell nuclei were stained with Hoechst 33342 (blue). (f) Real time quantitative RT-PCR of the ngdf mRNA level in neurons from T0 (15 min post-lesion, black) vs. 24 h post-lesion (gray) CNS. A leech 18 S ribosomal RNA was used as internal reference. Significance (*p < 0.05, **p < 0.01 vs. T0) was calculated by paired T-test (bar represents Standard Errors of Mean). (g) Chemotactic effect on microglia cells of conditioned medium (C.M.) from primary culture of microglia (light gray) or neurons (dark gray). The chemotactic effect of C.M. from neurons was also assessed on anti-ALK5-incubated microglia to specifically neutralize ALK4/5 in microglia (dark gray). The chemotactic effect of anti-TGF-β-incubated C.M., specifically neutralizing neurons-derived nGDF, was also evaluated on microglia (dark gray). A control medium was used as negative control (black). Significance (*p < 0.05, **p < 0.01, ***p < 0.001) was calculated by ANOVA Dunnett’s multiple comparisons test (bar represents Standard Errors of Mean).
