Kod plus kit

T4 DNA ligase and Lipo8000 transfection reagent were purchased from Beyotime Biotechnology (Shanghai, China). 2 × Taq Plus Master Mix (Dye Plus) and T4 polynucleotide kinase was purchased from Vazyme Biotechnology (Nanjing, China). The KOD-Plus Mutagenesis Kit and KOD-Plus Kit were from TOYOBO (Osaka, Japan). Ni²⁺-NTA Superflow resin was purchased from Qiagen Inc. (Hilden, Germany). Dynabeads Protein G, 4,6-diamidino-2-phenylindole (DAPI), MitoTracker, Pierce Silver Stain kit, and Alexa Fluor 488-conjugated secondary antibody were obtained from Thermo Scientific (Waltham, MA, USA). [¹⁴C]Thr, [¹⁴C]Ser and [¹⁴C]Tyr were obtained from Perkin Elmer Inc. (Waltham, MA, USA). PrimeScript RT Master Mix and PrimeSTAR Max DNA Polymerase were obtained from TaKaRa (Kyoto, Japan). Trelief Prestained Protein Ladder and oligonucleotide primers were obtained from Tsingke (Shanghai, China). Competent E. coli BL21(DE3) were purchased from Weidi Biotechnology (Shanghai, China). Serum-free Cell Cryopreservation Medium was obtained from Epizyme Biomedical Technology (Shanghai, China). Anti-c-Myc magnetic beads, kanamycin sulfate and ampicillin sodium were purchased from MedChemExpress (MCE, New Jersey, USA).

Small pieces of a colony (3 × 3 mm) grown on malt extract agar (MEA) medium at 25 °C for 10 d were put into 2-mL Cryo tubes. DNA was extracted using the PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). PCR was performed using a KOD-Plus Kit (Toyobo, Osaka, Japan) following the manufacturer’s protocol. The rDNA large subunit region (LSU D1/D2) was amplified with the primer pair NL1/NL4 (O’donnell, 1993 ). To amplify the ITS region, the primers ITS1 and ITS4 were used (White, Bruns, Lee, & Taylor, 1990 ). Amplification of the DNA fragments was performed using the GeneAmp PCR System 9700 (Applied Biosystems). PCR products were checked by agarose gel electrophoresis and purified using an AMPureKit (Agencourt Biosciences, Beverly, MA, USA). Sequencing reactions were performed by using the Big Dye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems) and the same PCR primers. The newly generated sequence data were deposited in the DNA Data Bank of Japan (DDBJ) under the accession numbers provided in Table 1.

Total RNA from adult C57/BL6 mice brains was prepared using the TRIzol reagent, according to the manufacturer's protocol (cat. no. 15596-026, Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and a glass homogenizer, this was incubated subsequently with DNase I for 1 h at 37°C to remove any contaminating DNA. RNA was reverse transcribed to cDNA using a M-MLV RTase cDNA Synthesis kit (cat. no. D6130, Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. The polymerase chain reaction (PCR) amplification of ADAM10 cDNA was performed using the KOD-Plus kit (Toyobo Co., Ltd., Osaka, Japan) and the following primers, sense, 5′GGTGAAACGCATAAGAATC-3′ and antisense, 5′CACTGAACTGCTTGCTCC-3′ under conditions as follows: Denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 40 sec, and extension at 68°C for 90 sec; and extension at 68°C for 5 min. The amplified PCR fragments were analyzed on agarose gels. The identity of the PCR products was confirmed with restriction analysis. For restriction reactions, the electrophoresed PCR products were purified from agarose gels using QIAquick Gel Extraction kit (Qiagen GmbH, Hilden, Germany) and cloned into pGEM-T Easy plasmid (Promega Corporation, Madison, WI, USA) following the manufacturer's protocol.