Fig. 1.

ADAM13 binds to Fz4 and Fz4-v1. (A) Co-immunoprecipitation experiments of Fz4 and Fz4-v1 with ADAM13. HEK293T cells were transfected with the various constructs cloned into the pCS2+ vector alone or in combination. ADAM13 was immunoprecipitated using the cytoplasmic domain antibody g821, and Myc-tagged (mt) Fz4 constructs were detected with mAb 9E10. Fz4-mt and Fz4-v1-mt co-precipitate with ADAM13 only when both proteins are co-transfected. Bands observed in lanes 2 and 3 of the total extract blotted with ADAM13 are non-specific. (B) Schematic diagram of mature ADAM13 including the metalloprotease (M), disintegrin (D), cysteine-rich (C), EGF repeat (E) and the cytoplasmic (Cy) domain. In co-immunoprecipitation experiments using a rabbit polyclonal antibody to the Fz4-v1 protein, all ADAM13-mt constructs containing the cysteine-rich domain were co-precipitated, while the disintegrin domain alone was not (D). (C) Real-time PCR on dissected CNC and stage-matched whole embryos (Total), detecting the level of Slug, Fz4, or Fz4-v1 expression, represented as a percentage of GAPDH expression. The average cycle threshold (CT) values in the CNC were as follows (Slug 24.7, Fz4 28.3, Fz4-v1 29.7, GAPDH 23.9). No CT was seen for water or no RT control. Error bars represent ±s.e.m. (D) Double in situ hybridization using short probes recognizing Fz4 or Fz4-v1 (blue) as well as probes recognizing the two CNC markers Sox10 and Twist (both red). The arrowheads point to the tip of the CNC segment. Scale bar: 500 µm. In situ hybridization for ADAM13 is presented as a reference marker (A13).