Ligases

The cDNAs of IFITM1, IFITM2, IFITM3, and ABHD16A from humans, pigs, and mice were synthesized from the isolated total RNA of Homo sapiens embryonic kidney HEK293 cells, porcine kidney epithelial PK15 cells, and mouse fibroblast 3T3 cells, respectively, using specific primers (see Table S1 in the supplemental material). To generate 3×Flag-, MYC-, S-tag-, VN173-, VC174-, DsRed-, and GFP-tagged IFITMs or ABHD16A, the cDNA fragments were digested with EcoRI and BamHI and individually cloned into the same sites of pcDNA3.1(+), pEYFP-N1, pDsRed-monomer-N1 and pEGFP-N1 vector, respectively. To knock down sABHD16A in PK15 cells, swine sabhd16a (GeneID 100155979) targeting sequence (5′-CGGCTGGTGGAAGAGTGTAAT-3′ was cloned into pLKO.1-TRC-Puro vector through AgeI and EcoRI sites. The primary vectors were purchased from Invitrogen (CA, USA). Restriction endonucleases, ligases, and other molecular biological reagents were obtained from TaKaRa (Japan).

Takara PCR Thermal Cycler Dice Gradient was used for DNA amplifications. PrimeSTAR GXL DNA polymerase (Takara Bio Inc., 1 μL), 5× PrimeSTAR GXL buffer (10 μL), dNTP Mixture (2.5 mM each) (4 μL), forward primer (10 pmol), reverse primer (10 pmol), and template (10 ng) were used after filling up to 50 μL with sterilized distilled water. PCR was done for 15 cycles of (98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1.5 min). DNA manipulation reagents such as restriction enzymes and ligases were purchased from Takara Bio Inc. and TOYOBO. To confirm nucleotide sequences, pET upstream primer (5′-ATGCGTCCGGCGTAGA-3′), duetdown1 primer (5′-GATTATGCGGCCGTGTACAA-3′), duetup2 primer (5′-TTGTACACGGCCGCATAATC-3′), T7 terminator primer (5′-GCTAGTTATTGCTCAGCGG-3′) were used.