Cloning and sequencing of the Danio rerio α3(IV)NC1 and characterization of recombinant Danio rerio α3(IV)NC1 domain. (A) Nucleotide and peptide sequence of the NC1 domain of Danio rerio type IV collagen α3-chain, (zα3(IV)NC1). The 2 regions homologous to Goodpasture epitope, E_A and E_B, are boxed. The conserved methionine and lysine (diamonds) residues, potentially involved in a conserved novel nondisulfide covalent crosslink,²⁷  are shown. The circles indicate the 12 conserved cysteines. (B) FLAG-tagged Danio rerio α3(IV)NC1 was recombinantly produced and an expected band at 29.4 kDa is recognized by the anti-FLAG antibody (lane 1), as well as with the anti-human α3(IV) NC1 antibody (lane 2). However, no binding with Goodpasture sera is observed (lane 3), despite prolonged exposure of film. (C) Native Danio rerio kidney extract (30 μg per lane) digested with bacterial collagenase was run on 15% SDS-PAGE under both reducing and nonreducing (NR) conditions. Under nonreducing conditions, only dimers (D) and trimers (T) can be observed with the anti-human α3(IV) NC1 antibody antibody (lane 1). Under reducing conditions, native α3(IV)NC1 monomers (M) are observed at the expected molecular weight (lane 3). FLAG-tagged recombinant human α3(IV)NC1 (rh-α3(IV)NC1; 250 ng) was used as positive control (lane 2).³⁵  (D) No reactivity with Goodpasture sera with native Danio rerio kidney extract can be seen (lane 1). FLAG-tagged rh-α3(IV)NC1 was used as positive control (lane 2).