Fig. 4. Multimerization is required but not sufficient for Shh early striatal neuronal differentiation-inducing activity. Gel filtration of Shh allows the classification of Shh multimers into three distinct size groups: >669 kDa (L, large), 669-69 kDa (M, medium), and monomers. Different N- and C-terminal lipid-containing proteins from cell culture supernatants derived from C17 cell lines (Fig. 3A) were affinity purified on a 5E1 anti-Shh antibody column. Affinity-purified proteins were separated by Superdex 200 gel filtration column chromatography (AKTA FPLC). Fractions eluted from the column were separated by SDS-PAGE, and probed with anti-Shh antibody by western analysis. (A) wtShh (N+C lipid), (B) C24S-Shh (C lipid alone), (C) ShhN (N lipid alone), (D) C24S-ShhN (no lipid). The elution profile of standard proteins on the Superdex column (Pharmacia) is indicated at the top of each gel. The fraction number is indicated at the bottom of each lane. SDS-PAGE standards are indicated on the left. (E-H) A comparison of the early striatal neuronal differentiation-inducing activities of multimeric wtShh, multimeric C24S-Shh, multimeric ShhN and monomeric ShhN. Rat E11 telencephalic explants were treated with multimeric wtShh (E; fractions 37 and 38; 8/8 explants lacked Dlx- or Islet1/2-expressing cells), multimeric C24S-Shh (F; fractions 37 and 38; 7/8 lacked Dlx- or Islet1/2-expressing cells), multimeric ShhN (G; fractions 37 and 38; 6/7 explants contained Dlx- and Islet1/2-expressing cells) or monomeric ShhN (H; fractions 59 and 61; 6/7 explants lacked Dlx- or Islet1/2-expressing cells). Anti-Dlx was detected with an anti-rabbit Cy2-conjugated antibody (green), and anti-Islet1/2 was detected with an anti-mouse Cy3-conjugated antibody (red).