Pcmv sirt1 t1 flag

SIRT1 cDNA was made from pCMV-SIRT1-t1-Flag (purchased from Sino Biological) via PCR amplification. SIRT1 cDNA was cloned into the FUGW vector using Seamless Cloning Kit (Beyotime, D7010M) and confirmed by DNA sequencing. HMC3 cells were plated into 24-well or 6 cm dish at appropriate intensity and cultured overnight. We transfected 150 ng plasmid per well into 24-well or 1.5 μg plasmid per well into 6 cm dish. SIRT1 plasmid or FUGW plasmid was transfected using ViaFect reagent (Promega, #E4981) according to manufacturer's instructions. After transfection for 24 h, cells were stimulated by Aβ for 72 h. The knockdown of SIRT1 was performed by the transfection with specific siRNA (Tsingke Biotechnology Co., Ltd.) using ViaFect reagent. The cloning primers used were as follows: SIRT1 (forward: 5′-TGGGCTGCAGGTCGACTCTAGAATGGCAGATGAAGCAGCTCTC-3′ and reverse: 5′-TTG ATATCGAATTCTAGACTATGATTTGTTTGATGGATAGTTCATGTCT-3′). The siRNA primers were as follows: siSIRT1-1 (forward: 5′-CACCUGAGUUGGA UGAUAUTT-3′ and reverse: 5′-AUAUCAUCCAACUCAGGUGTT-3′) and siSIRT1-2 (forward: 5′-GUCUGUUUCAUG UGGAAUATT-3′and reverse: 5′-UAUUCCACAUGAAACAGACTT-3′).

SIRT1 cDNA was generated from pCMV-SIRT1-t1-Flag (purchased from Sino Biological) via PCR amplification and then cloned into the FuGW vector by using Seamless Cloning Kit (Beyotime, D7010M) according to the manufacturer’s instructions and confirmed by DNA sequencing. Cells were transfected with constructed SIRT1 plasmid using lipofectamine 3000 (ThermoFisher Scientific, L3000001). After transfection for 24 h, cells were stimulated by Aβ. The knockdown of SIRT1 was performed by the transfection with specific siRNA (Tsingke Biotechnology Co., Ltd.) using lipofectamine 3000.
The cloning primers were as follows:
Forward, TGGGCTGCAGGTCGACTCTAGAATGGCA GATGAAGCAGCTCTC;
Reverse, TTGATATCGAATTCTAGACTATGATTTGTTTGA TGGATAGTTCATGTCT;
The siRNA primers were as follows:
siSIRT1-1: Forward, CACCUGAGUUGGAUGAUAUTT;
siSIRT1-1: Reverse, AUAUCAUCCAACUCAGGUGTT;
siSIRT1-2: Forward, GUCUGUUUCAUGUGGAAUATT;
siSIRT1-2: Reverse, UAUUCCACAUGAAACAGACTT;