Pcmv n myc

The full-length cDNAs of CgVDAC2 and CgBak were sub-cloned using In-Fusion HD Cloning Kit (TaKaRa, Shiga, Japan) into the mammalian expression vectors pCMV-N-Myc and pCMV-N-Flag (Beyotime, Jiangsu, China), respectively, according to the manufacturer’s instructions. To investigate the subcellular localization of CgVDAC2, the pEGFP-N1-CgVDAC2 plasmid was constructed using In-Fusion HD Cloning Kit (TaKaRa, Shiga, Japan). The pEGFP-N1-CgBak plasmid was also constructed using the same kit for the overexpression experiments in HEK293T cells. In addition, CgVDAC2 and CgBak were sub-cloned into the two-hybrid plasmids pGADT7 and pGBKT7 (TaKaRa, Shiga, Japan), respectively, and used in the yeast two-hybrid system.

Total RNA of the mouse testis was extracted using TRIzol Reagent (Invitrogen), and cDNA was generated using a PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Full-length mice Sun1 (NM_024451), Terb1 (NM_180958), Terb2 (NM_028914), Majin (NM_001165919), Spdya (NM_029254), Cdk2 (NM_183417), Cyclin E1 (NM_007633) and E2 (NM_001037134) were amplified by PrimeSTAR Max DNA Polymerase (TaKaRa) from cDNA using primers containing specific restriction sites. The genes were cloned into expression plasmids p3 × FLAG-myc-CMV-24 (Sigma, St. Louis, MO, United States), pEGFP-C1 (Clontech, Mountain View, CA, United States), pmCherry-C1 (Clontech), and pCMV-N-Myc (Beyotime Biotechnology, Shanghai, China). Truncated mutants were generated by PCR, and deletion mutants were generated using overlapped primers with mutations. The primers were produced by GENEWIZ (Suzhou, China). All plasmids were verified by Sanger sequencing (GENEWIZ).