In our current study, we constructed a eukaryotic expression vector containing the PHD3 gene and detected its expression in human hepatoma Lazertinib order cell line (HepG2) cells to establish a foundation for future studies. Materials and methods Materials Plasmid pcDNA 3.1(+) was obtained from the Central Laboratory of Affiliated Hospital of Guangdong Medical College (Guangdong, China). E. coli DH5α was gained from the Pathogenic Biology Laboratory of Guangdong Medical College. Human hepatoma cells (HepG2) were obtained from the Laboratory of Hepatobiliary
Surgery. Placenta tissue and the written informed consent for this tissue were obtained from the Operating Room of Affiliated Hospital of Guangdong Medical College. RNAiso Plus, High Fidelity Prime Script™ RT-PCR Kit, TaKaRa Agarose Gel DNA Purification Kit Ver.2.0, DL10,000 DNA Marker, DNA A-Tailing Kit, pMD19-T Simple Vector, DNA Ligation Kit Ver.2.0, Hind III, check details Xho I, TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0 and SYBR® Prime Script® RT-PCR Kit II (Perfect Real Time) were purchased from TAKARA (Japan). Neonatal Bovine Serum was
acquired from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd (China). Dulbecco’s modified Eagle’s medium(DMEM)was purchased from Hyclone Company (USA). www.selleckchem.com/products/AC-220.html Lipofectamine™ 2000 was purchased from Invitrogen Biotechnology (USA). DMSO was purchased from Sigma (USA). 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT) was purchased from Sangon Biotech (Shanghai) Co., Ltd (China). Primary rabbit polyclonal anti-EGLN3 antibody was purchased from Jiamay Biotech Company (China). Primary rabbit polyclonal anti-Caspase-3 antibody was purchased from Zhongshan Goldenbridge Biotechnology Oxaprozin CO., LTD (China). Primary rabbit polyclonal anti-tubulin antibody, a BCA protein assay kit and BeyoECL Plus were purchased from Beyotime Institute of Biotechnology (China). Vector construction Total RNA extraction
and PHD3 cDNA synthesis Total RNA from placental tissue was extracted with RNAiso Plus according to the manufacturer’s instructions. First, 1 μg of total RNA was used to synthesize full-length PHD3 CDS with High Fidelity Prime Script™ RT-PCR Kit. A pair of specific primers, containing Hind III and Xho I restriction enzyme cutting sites, were designed: forward 5′-CCCAAGCTTGATGCCCCTGGGACACATCAT-3′ and reverse 5′-CCGCTCGAGTCAGTCTTCAGTGAGGGCAGA-3′. Purification of PHD3 cDNA and ligation with pMD19-T simple vector The RT-PCR products were separated with 1.5% agarose gel electrophoresis, and the target fragments were retrieved and purified by TaKaRa Agarose Gel DNA Purification Kit v.2.0. The target fragments were polyadenylated using DNA A-Tailing Kit; these fragments were then ligated into pMD19-T Simple Vector with DNA Ligation Kit v.2.0 (TA Clone). The recombinant pMD19-T-PHD3 was transformed into E. coli DH5α competent cells for amplification. Recombinant vectors were isolated from transformants by TaKaRa MiniBEST Plasmid Purification Kit v.2.