Characterization of zebrafish Padi2. (A) Schematics of padi2 transcripts, with exons represented by solid boxes and introns by connected lines (slashes indicate shortening of relative length for display purposes). Left: A list of the corresponding last seven digits of Ensembl ID from GRCz11 and GRCz10 genome assemblies (full Ensemble IDs listed in Materials and methods). Right: A list of the names based on GRCz10 used to reference the transcripts. Cloned transcripts discussed in this paper are in green, and arrows highlight exon 10. (B) Full amino acid sequences of human PAD2 and predicted zebrafish Padi2 splice variants (201a and 202). Amino acids are highlighted (as indicated in key) to demonstrate calcium binding, catalytic residues, and substrate-binding residues. Black arrowheads indicate amino acids referred to in F and Fig. 1 B. (C) Full Western blot (from Fig. 1 F) of pooled larvae probed with antibodies against zebrafish Padi2 and actin. WT and padi2^−/− lysates were probed. Arrow demonstrates expected size of Padi2 transcripts at ∼75 and 80 kD, and asterisk marks ∼200 kD species. Notably, this antibody did not detect a protein of equivalent size to the predicted transcript 203 (∼35 kD). Representative blot from four replicates. (D) Western blot of pooled WT larvae probed with preimmune serum and actin antibody. (E) zPadi2 Western blot of pooled 2 dpf larvae. Lane 1, WT; lane 2, padi2^−/−; lane 3, padi2 201a mRNA-injected padi2^−/− larvae. (F) Citrullination activity of Padi2 202 and individual point mutations in select calcium-binding and catalytic amino acids (colors correspond to highlighted residues in B). Fold change of enzymatic activity normalized to WT Padi2 202. Data represent two independent experiments, and WT values are also represented in Fig 1 A.