Pcdna3.1 hygro vector

The ORF of IE2 was first amplified from cDNA of infected HFF (24 h p.i.) using an iProof polymerase kit (Biorad). Restriction sites (BamHI/XbaI containing primers) were added in a second PCR step to the resulting amplicon for subsequent insertion into the pcDNA3.1/Hygro(+) vector (Thermofisher Scientific). Sequence identity of the insert with the AD169 IE2 CDS (NCBI FJ527563.1) was confirmed by Sanger sequencing of both the forward and reverse strand.
HEK293 cells were transfected with 850 ng of pcDNA-IE2 or empty vector control plasmid in 24-well plates using 2 μL Turbofect (Thermofisher Scientific) according to the manufacturer’s instructions. Two days post transfection, lysates were harvested for analysis via Western blot.

Open reading frames encoding C11orf94, Izumo1, SOF1, SPACA6, and ACE3 were amplified by PCR from murine testis cDNA and equipped with C-terminal 3xFLAG, (3x)myc, or HA tags, respectively. Coding sequences were inserted into the pcDNA3.1 hygro⁺ vector (Thermo Fisher Scientific) using Hind III and Xho I (C11orf94, SPACA6, and Izumo1) or Bam HI and Xho I (SOF1 and ACE3) restriction sites. For analysis of the membrane topology of C11orf94, an artificial NIS motif was added to the N or C terminus of the protein, respectively. Plasmids encoding NotchΔE-eGFP (53 (link)), mSPPL2c-myc, and mSPP-myc and the catalytically inactive mSPPL2c D457A variant (20 (link)) have been described previously. The mutations D395A and F455L were introduced into the SPPL2c-coding sequence by site-directed mutagenesis, and the resulting open reading frames were ligated into the pcDNA hygro⁺ expression vector using Bam HI and Not I sites. For propagation of plasmids, Escherichia coli XL1-Blue cells (Stratagene) were used.

The Tas2r108 (NM_020502.1) and Tas2r126 (NM_207028.1) sequences with the bases that encode 45 amino acids of rat somatostatin receptor 3 [22 (link)] and HSV glycoprotein epitope tag attached at their 5′ and 3′ ends, respectively, were cloned into a pcDNA™3.1/Hygro(+) vector (Thermo Fisher Scientific Inc., Waltham, MA, USA). The vector was transfected into 3T3-L1 preadipocytes using Lipofectamine 3000 reagent (Thermo Fisher Scientific Inc.) and Opti-MEM™ I reduced serum medium (Thermo Fisher Scientific Inc.). Three days after transfection, hygromycin B (0.5 mg/mL) was added to the medium and the cells were selected for 1 week. The selected cells were used for the experiments.

A pNIS-FL2-TurboFP635-pCMV-Rluc plasmid DNA expressing the reporter gene driven by dual promoters was established by Cosmo Genetech Co. Ltd. (South Korea) using pNIS-FL2-TurboFP635 and pcDNA3.1/Hygro (+) vectors. pNIS-FL2-TurboFP635 vector was a kind gift from Dr. Abhijit De, ACTREC, India. pcDNA3.1/Hygro (+) vector was purchased from Invitrogen (Carlsbad, CA, USA). For monitoring NIS promoter/enhancer, the downstream region (clockwise) of NIS promoter included the gene for firefly luciferase (Fluc) and TurboFP635. Cytomegalovirus promoter with Renilla luciferase (Rluc) gene was placed in the opposite direction (anti-clockwise) of NIS promoter region to evaluate cell viability. The details of vector diagram are indicated in Supplementary Figure 1.