Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. OmPRLR1 and OmPRLR2 nucleotide and deduced amino acidic sequences. Cysteines are highlighted with gray background, N-glycosylation sites are underlined, WS domain is printed in bold and underlined, transmembrane region is italicized and dotted-underlined, Box 1 is highlighted with black background, Box 2 is highlighted with gray background, and tyrosine phosphorylation sites are boxed with tyrosine residues printed in bold. Ubiquitination motif is dotted-underlined and 14-3-3 binding motif is double-underlined.

• Supplemental Figure S2 -

  Fig. S2. Multiple sequence alignment of PRLR1 and PRLR2. Conserved cysteine residues are identified with arrowheads. The WS motif, transmembrane region, Box 1 and Box 2, ubiquitination motif, 14-3-3 binding motif and conserved tyrosine phosphorylation motifs are labeled. Om: Oreochromis mossambicus (Mozambique tilapia, PRLR1: EU999785; PRLR2: EU999783), Ga: Gasterosteus aculeatus (three-spined stickleback, PRLR1: ENSGACP00000021749; PRLR 2: ENSGACP00000018184), Tr: Takifugu rubripes (spiny pufferfish, PRLR1: Fugu484Sc44; PRLR2: BAE94535 (type2), Tn: Tetraodon nigroviridis (green pufferfish, PRLR1: Chr4 long AY374480; PRLR2: AY374409modif), Ca: Carassius auratus (crucian carp, PRLR1: AAF23268), Cc: Cyprinus carpio (common carp, form a PRLR1: AAK95833), Dr: Danio rerio (zebrafish, PRLR1: AAQ84555), As: Acanthopagrus schlegelii (PRLR2: ABO61870), Sa: Sparus aurata (gilthead seabream, PRLR2: AAG17629), Km: Kryptolebias marmoratus (PRLR2: ABK97641).

• Supplemental Figure S3 -

  Fig. S3. Controls for antibody specificity. Lanes were loaded as follows: (1) PRLR immune serum; (2) pre-absorption with 100ng ml⁻¹ antigenic peptide (SESSSEESSEKTKSSQ) overnight at 4°C followed by PRLR immune serum; (3) pre-immune serum, 1:500 dilution; (4) no primary antibody. Primary antibody incubations were done overnight at 4°C at 1:500 dilution in Tris-buffered saline containing 5% non-fat dry milk (TBSM) for conditions 1−3 (for condition 4 only TBSM was used). Secondary antibody incubations (1:10,000 dilution for 1 h at room temperature), blocking, loading (20 µg total protein from fresh water tilapia gill), all washing steps, and Supersignal Femto (Pierce) development were identical for all four conditions. Membranes were cut before separate primary antibody incubations and re-assembled before imaging with a ChemiImager (AlphaInnotech).