FIGURE 3.

HNK-1 epitopes were carried by TNC in NSCs. a, major HNK-1-carrier protein in secondary neurospheres (arrow) was immunoprecipitated with control mouse IgG or anti-HNK-1 antibody (IP) and then detected by silver staining. By LC-MS/MS analysis, the major HNK-1-carrier protein was identified as TNC. b, amino acid sequence of TNC is shown. The sequences of observed peptides are shown in green. The putative signal sequence is shown in blue. Twenty potential N-glycosylation sites are shown in red. The amino acid sequence of the alternatively spliced domain C is underlined. c, proteins from the secondary neurospheres were immunoprecipitated with control mouse IgG or H300 anti-TNC antibody (IP) and then subjected to Western blot analysis with anti-HNK-1 antibody. d, NSCs prepared from primary neurospheres were treated with PBS containing 3% fetal bovine serum and 0.1% Triton X-100 and then stained with anti-TNC and anti-HNK-1 antibodies as primary antibodies, and DyLight488-conjugated anti-rat IgG antibody (green) and Cy3-conjugated anti-mouse IgG antibody (red) as secondary antibodies. Nuclei were stained with 2 μg/ml Hoechst 33258 (blue).