BEX4 Functional Analysis via Genetic Manipulations

Full-length human BEX4 was generated by PCR. Full-length human BEX4 was also subcloned into pGEX-KG (GST-BEX4) and pTAP (TAP-BEX4) for the GST pull-down assay and tandem affinity purification (TAP), respectively. Fragments encoding BEX4 were subcloned into pEGFP-C1 (Clontech, Mountain View, CA, USA) to generate a GFP-fused BEX4 expression vector (pEGFP-BEX4). BEX4 mutant alleles were generated by site-directed mutagenesis. Comlementary DNAs of the BEX4 wild-type (WT) and five mutant versions, in which a single serine or single threonine residue was exchanged for alanine (S3A, T29A, S35A, T94A, and T107A), were subcloned into pEGFP-C1 and pGEX-KG. The pCMV-HA-PLK1 plasmid has been published previously^{18 (link)}. To specifically knock down BEX4, short hairpin (sh)RNAs were cloned into pSuper puro (Oligoengine, Seattle, WA, USA) containing the H1 promoter according to the manufacturer’s instructions. Oligonucleotides encoding a shRNA against BEX4 (5′-GGCCATACCTAATAGGCATAT-3′), human CDK1 (5′- GGGGATTCAGAAATTGATC-3′), human PLK1 (5′-GGTCCATTGGGTGTATTCATGT-3′) or the luciferase control (5′-CATACGCGGAATACTTCGA-3′) were synthesized, and the efficiencies of each shRNA were determined by immunoblotting^{12 (link),18 (link),19 (link)}. For transient transfection, the cells were electroporated using a microporator (Digital Biotechnology, Seoul, Korea).