Human fetal brain cdna library

The yeast two-hybrid screening was performed using a human fetal brain cDNA library (Clontech) in the reporter strain AH109 using the second and third PDZ domains of human Par3 (amino acid residues 408–626) as a bait, as previously described (Izaki et al, 2005 (link); Iwakiri et al, 2013 (link)). Among 1.8 × 10⁶ clones screened, a positive clone obtained encodes the intracellular region of TMEM25 (amino acid residues 276–366) as revealed by sequencing analysis. For analysis of the interaction between TMEM25 and Par3, HF7c cells were transformed with combinations of pGADGH encoding TMEM25 and pGBK encoding Par3. After selection for the Trp⁺ and Leu⁺ phenotypes, transformed cells were tested for their ability to grow on plates lacking histidine supplemented with 5 mM 3-aminotriazole.

The Matchmaker GAL4 Two-Hybrid System 3 (Clontech) was used for the screening of a human fetal brain cDNA library (Clontech) with pGBKT7-HBc as bait. Co-transformants were selected on synthetic dropout (SD), media lacking leucine, and tryptophan (SD/-Leu/-Trp) and were validated by growth on SD, media lacking leucine, tryptophan, adenine, and histidine (SD/-Leu/-Trp/-Ade/-His) and containing 5-bromo-4-chloro-3-indolyl-α-D-galactoside (X-α-gal). Then, positive colonies were sequenced (Invitrogen, Carlsbad, CA, USA).

The construct UL80.5/UL86 was derived from the DNA of Towne-BAC [5 (link)], while the construct UBC9 and the constructs SUMO1/SUMO2/SUMO3 were extracted from the human fetal brain cDNA library (Clontech, Mountain View, CA, USA). UL80.5 cDNA was sub-cloned into pGBKT7 and pCMV-HA vectors. UBC9 cDNA was sub-cloned into pGADT7, pCMV-Myc, and pRK11-Flag vectors (provided by Dr. Hongbin Shu, Wuhan University, China). Truncated UBC9 mutants were generated by PCR using pGADT7-UBC9 as the template and then inserted into NdeI/BamHI digested pGADT7. SUMO1 cDNA was sub-cloned into pCMV-Myc and pLenti vectors. SUMO2 and SUMO3 cDNA were sub-cloned into pCMV-Myc vectors. The constructs that contained the sequences that encoded different UL80.5 (K-R) mutants were generated by a Fast Mutagenesis kit (Transgen Biotech, Beijing, China) using pCMV-HA-UL80.5 as the template with primers that contained appropriate nucleotide change (Table 1). There were a total of ten lysine residues that individually mutated to arginine which were separately called K41R, K163R, K175R, K178R, K205R, K208R, K242R, K296R, K355R, and K371R. The construct that contained the sequences encoding the UBC9 (C93S) mutant was also generated by a Fast Mutagenesis kit using pRK11-FLAG-UBC9 as the template.

The open reading frame region of the human neuritin gene was amplified with PCR and was cloned into pGBK-T7 (Clontech) to generate the bait plasmid pGBK-T7-neuritin. A human fetal brain cDNA library (Clontech) was screened with the pGBKT7-neuritin bait plasmid according to the manufacturer’s instructions. All of the positive clones were repeatedly screened on the synthetic nutrition drop-out culture medium plates. After initial clustering on positive colonies with PCR and electrophoresis, identified prey cDNA-encoding proteins were sequenced and the sequence alignment was analyzed.

The TIPRL cDNA (acess number NP_690866.1) was amplified from a human fetal brain cDNA library (Clontech) and cloned into pET-TEV, a modified pET28a vector harboring an N-terminal hexa-histidine tag followed by a TEV cleavage site. The TIPRLΔN construct was obtained by amplifying the cDNA from pET-TEV-TIPRL using a forward primer that resulted in a 15-residue N-terminal deletion. The resulting PCR product was subcloned into pET-TEV. The mutants were obtained by thermal cycling with Pfu (Thermo Scientific) and digestion of parental DNA with DpnI (Thermo Scientific), followed by transformation into DH5α and sequencing. The sequences of primers used for mutagenesis are shown in Table S2.