Pcdna4 v5

An open reading frame of Pep51 was amplified using gene-specific primers with extensions to vector ends (Fw; 5’-CCGGAATTCGCCATGTTGAGCATAAAATC-3’, and Rv: 5’- AAAGCGGCCGCTTAGCACAGTTTATTTG-3’, including stop codon), which contain an EcoRI site and NotI site, respectively. The amplified PCR product and the expression vector pcDNA4/V5 (Thermo Fisher Scientific, Waltham, MA, USA) were digested with EcoRI and NotI restriction enzymes, followed by ligation using Takara Ligation Mighty Mix (Takara, Shiga, Japan) according to the manufacturer’s instructions. The expression vector, empty pcDNA4 vector (0.1 μg), or transfection medium alone was transiently transfected into 5×10⁴ African green monkey kidney fibroblast cells (COS-7 line) or genetically engineered human embryonic kidney cells (HEK293MSR line) in 9.5-mm wells of multi-well glass-bottom dish (Matsunami, Osaka, Japan) with Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. COS-7 cells and HEK293MSR cells were grown under 5% CO₂ at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, and DMEM supplemented with 10% heat-inactivated FBS and 1% non-essential amino acids, respectively. Transfection efficiency (approximately 60-70%) was evaluated by an immunocytochemistry as described below.

Open reading frames (ORFs) of EP2, CTR, and oxytocin receptor (OXTR) were amplified from the OV3121 by RT-PCR. The ORF of EP2 was subcloned in frame into 5’-end of the pcDNA4/V5 (Thermo Fisher Scientific) and pAmCyan1-N1 cyan fluorescent protein (CFP) vector (Clontech, Kyoto, Japan) at the EcoRI/XhoI and NheI/XhoI, sites, respectively. The CTR was subcloned into the pcDNA4/V5 and pZsYellow1-N1 yellow fluorescent protein (YFP) vector (Clontech) at the NheI/XhoI sites. The OXTR was subcloned into the pAmCyan1-N1 CFP vector at the EcoRI/NheI sites. Each construct was transiently transfected into HEK293MSR cells using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions.