Supplementary Figures,Table, and Captions

Supplementary figures,table, and captions

Fig. S1

Caption Fig. S1: Total RNA was extracted from gills, stomach, intestine, skin, kidney, head kidney, spleen, muscle, heart, brain, blood, and liver of healthy cobia, respectively. RNA sample concentration was assessed with a NanoDrop spectrophotometer prior to the reverse transcription (RT). The first-strand cDNA was synthesized from 2 μg of DNase-treated total RNA. The same amount cDNA was used as template for PCR reactions. RT-PCR was used to detect the expression levels of β-actin and RcCC2 mRNA in various tissues of non-stimulated cobia. M, DL2000 DNA marker; Gi, gills; St, stomach; In, intestine; Sk, skin; Ki, kidney; Hk, head kidney; Sp, spleen;

Mu, muscle; He, heart; Br, brain; Bl, blood; Li, liver.

Fig. S2 （A）

（B）

（C）

Caption Fig. S2: Total RNA was extracted from liver of cobia prior to injection (0 h) and at 3, 6, 12, 24, 48, and 72 h post-injection. RNA sample concentration was assessed with a NanoDrop spectrophotometer prior to the reverse transcription (RT). The first-strand cDNA was synthesized from 2 μg of DNase-treated total RNA. The same amount cDNA was used as template for PCR reactions. RT-PCR was used to detect the expression levels of β-actin and RcCC2 mRNA in liver at various times after injection with PBS (A), poly(I:C) (B) or inactivated bacterial vaccine (C).

Fig. S3 Caption Fig. S3:

The cDNA and deduced amino acid sequences of RcCC2 from cobia. The nucleotide sequences are on the upper lines and putative amino acid sequences are in the lower lines. The start and stop codon are boxed. The four conserved cysteines (C) are marked by triangles. The predicted cleavage of the signal sequence to the putative mature protein, two cytokine RNA instability motifs

(ATTTA) , and a polyadenylation signal (AATAAA) are underlined.

Table S1 Accession Species Chemokine Amino acid Accession Species Chemokine Amino acid numbers name Identities (%) numbers name identities (%) P22362 Human CCL1 18.0 DQ173276 Catfish SCYA101 15.3 P13500 Human CCL2 15.3 DQ173280 Catfish SCYA106 34.2 P10147 Human CCL3 14.4 DQ173286 Catfish SCYA112 20.7 P13236 Human CCL4 15.3 DQ173287 Catfish SCYA113 21.6 P13501 Human CCL5 15.3 DQ173289 Catfish SCYA115 15.3 P80098 Human CCL7 15.3 DQ173290 Catfish SCYA116 15.3 P80075 Human CCL8 15.3 DQ173293 Catfish SCYA119 22.5 P51671 Human CCL11 11.7 DQ173295 Catfish SCYA121 9.0 Q99616 Human CCL13 14.4 DQ173298 Catfish SCYA126 16.2 Q16627 Human CCL14 14.4 AAF23867 Rainbow trout CK1 19.8 Q16663 Human CCL15 16.2 AAM09300 Rainbow trout CK2 13.5 O15467 Human CCL16 14.4 AJ315149 Rainbow trout CK3 15.3 Q92583 Human CCL17 18.0 CA371157 Rainbow trout CK4A 18.9 P55774 Human CCL18 14.4 CA383670 Rainbow trout CK5A 14.4 Q99731 Human CCL19 22.5 CA378686 Rainbow trout CK9 11.7 P78556 Human CCL20 18.9 CA361535 Rainbow trout CK10 39.6 O00585 Human CCL21 24.3 CA346383 Rainbow trout CK12B 23.4 O00626 Human CCL22 16.2 ADE58983 Gilthead seabream CK1 18.9 P55773 Human CCL23 15.3 ADE58986 Gilthead seabream CK3 17.1 O00175 Human CCL24 17.1 ADE58984 Gilthead seabream CK5 14.4 O15444 Human CCL25 10.8 ADE58987 Gilthead seabream CK7 16.2 Q9Y258 Human CCL26 14.4 ADE58985 Gilthead seabream CK8 18.9 Q9Y4X3 Human CCL27 14.4 ADE58982 Gilthead seabream CK10 57.7 Q9NRJ3 Human CCL28 18.0 JF975591 Cobia RcCC1 17.1 P27784 Mouse CCL6 9.9 JF975592 Cobia RcCC3 18.0 P51670 Mouse CCL9 15.3 ABC69047 Turbot KC70 56.8 Q62401 Mouse CCL12 16.2 AEL31667 Half-smooth CsCCK1 43.2 tongue sole

Caption Table S1:

Amino acid identity of RcCC2 with mammals and other fish CC chemokines.