P3 flag cmv 14 expression vector

CIK cells were grown in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). GCRV-873 was propagated in CIK cells using MEM supplemented with 2% FBS. Plasmids used in this study, including the pTurboGFP vector (Evrogen) and p3×FLAG-CMV-14 expression vector (Sigma-Aldrich Co. LLC), were stored in our laboratory. vdra-FLAG, vdrb-FLAG, rxrga-FLAG, rxrgb-FLAG, and rxrbb-FLAG were obtained using the primer pairs vdra-F1/vdra-R1, vdrb-F1/vdrb-R1, rxrga-F1/rxrga-R1, rxrgb-F1/rxrgb-R1, and rxrbb-F1/rxrbb-R1, respectively, and were cloned into the p3×FLAG-CMV-14 vector. vdrb-GFP was obtained using the primer pairs vdrb-F2/vdrb-R2 and was cloned into the pTurboGFP-N vector. The primers used for plasmid constructs are listed in Table S1 in the supplemental material.

Based on zebrafish mRNA sequence (GenBank accession No: NM_194395), primers FLAG-BIRC2F/FLAG-BIRC2R were used for cloning the open reading frame (ORF) of zebrafish BIRC2, and inserted into the p3×FLAG-CMV ™-14 Expression Vector (Sigma-Aldrich). The primer sequences used for plasmid construction were listed in Table 1.
Domain architecture analysis was performed using Batch CD-Search (NCBI), and search results were visualized through the TBtools (20 (link)). Construction of phylogenetic tree was performed using the Neighbor-Joining method with 10,000 bootstrap replications using MEGA X software. Accession numbers of BIRC2 proteins from different species used are as follows: zebrafish (Danio rerio) BIRC2, XP_005161213.1; common carp (Cyprinus carpio) BIRC2, XP_018950329; channel catfish (Ictalurus punctatus) BIRC2, NP_001187106.1; rainbow trout (Oncorhynchus mykiss) BIRC2, XP_036845322.1; Japanese medaka (Oryzias latipes) BIRC2, XP_023818594.1; Atlantic cod (Gadus morhua) BIRC2, XP_030216815.1; spotted gar (Lepisosteus oculatus) BIRC2, XP_006627852; human (Homo sapiens) BIRC2, NP_001157.1; house mouse (Mus musculus) BIRC2, NP_031491.2; chicken (Gallus gallus) BIRC2, NP_001007823.1; green anole (Anolis carolinensis) BIRC2, XP_016848156.1; and common frog (Rana temporaria) BIRC2, XP_040196143.1.

JNK1 fusion constructs, K55R substitution mutants, and V5 tag-containing mutants were created using the PrimeSTAR Mutagenesis Basal Kit (Takara Biomedical) with zebrafish JNK1 cDNA and pcDNA6/V5-His A expression vector (Invitrogen), or derivatives, as the templates and the appropriate combinations of the forward and reverse oligonucleotide primers. An nSMase1-FLAG tag fusion construct and an S270A or S270E substitution mutant were generated using the PrimeSTAR Mutagenesis Basal Kit (Takara Biomedical) with zebrafish nSMase1 cDNA,^{16 (link)} p3 × FLAG-CMV-14 expression vector (Sigma-Aldrich), and pET-16b vector for the recombinant protein. The fidelity of the nSMase1 and JNK1 mutants was confirmed by sequencing. His-tagged recombinant proteins containing nSMase1 and various mutants were purified as described previously.^{16 (link)}