Figure 1.
Specificity of netrin antibodies. A, Peptide antigens were synthesized that correspond to sequences derived from the mature N terminus of chick netrin-1 (CN1) and chick netrin 2 (CN2). These sequences are unique to chick netrin-1 and chick netrin-2, respectively. Peptides also were synthesized that correspond to sequences within the domain V EGF-like loops (PN1, PN2) that are conserved in vertebrate netrin-1, netrin-2, and netrin-3 sequences. The PN1 peptide sequence is identical to the corresponding sequence of chick, rat, and mouse netrin-1 and contains a single conserved amino acid substitution in chick netrin-2. PN1 is poorly conserved in mouse netrin-3 and netrin-4, containing six of 20 and 17 of 20 amino acid substitutions, respectively. The PN2 sequence is identical in netrin-1 and netrin-2 in chick, rat, and mouse netrin-1. Two conservative amino acid substitutions are present in mouse netrin-3, and the corresponding sequence in mouse netrin-4 is poorly conserved, containing 10 of 18 amino acid substitutions. Recombinant chicken netrin-1 domain VI–V (PN3) is a 429 amino acid sequence that shares ∼90% amino acid identity with mouse netrin-1, ∼75% amino acid identify with chick netrin-2, ∼57% amino acid identity with mouse netrin-3, and ∼35% amino acid identity with mouse netrin-4. B, Antibody specificity was assessed by using purified recombinant netrin protein and Western blot analysis. In total, 50 ng of purified recombinant full-length myc-tagged chick netrin-1 (n-1) or chick netrin-2 (n-2) protein was loaded per lane. The 9E10 monoclonal antibody against a myc epitope tag confirmed that approximately the same amount of recombinant protein was loaded in each lane. Molecular weight markers, indicated with black bars on the left, correspond to 116, 97, and 66 kDa. Antibodies raised against peptide epitopes (CN1) and (CN2) are specific for chick netrin-1 and chick netrin-2, respectively. Antibodies raised against the conserved epitopes (PN2, PN3) recognize both recombinant chick netrin-1 and chick netrin-2.