Dna primers

The reagents and analysis systems used were as follows: heroin (purity 92.09%, provided by the Guizhou Public Security Bureau), pentobarbital sodium (Sigma, USA), naloxone hydrochloride (Chongqing YaoPharma, China), mouse monoclonal anti-SIRT1 (Abcam, USA), rabbit polyclonal anti-Cdk5, rabbit polyclonal anti-NF-κB (Bioss, China), rabbit monoclonal anti-FOXO1 (Abcam, USA), rabbit polyclonal anti-Cbp (Bioss, China), rabbit monoclonal anti-PSD95 (Abcam, USA), rabbit monoclonal anti-Syn (Abcam, USA), mouse monoclonal anti-beta-actin (Abcam, USA), SuperScript III RT reverse transcription kit (Invitrogen, USA) Sybr qPCR mix (Invitrogen, USA), Plasmid Mini kit (TransGen Biotech, Beijing, China), gel extraction kit (Omega), Trans2K Plus II DNA Marker (TransGen, Beijing, China), DNA primers (Invitrogen, USA), DNA sequencing (Invitrogen, USA), restriction endonuclease EcoRI (NEB, USA), T4 DNA Ligase (NEB, USA), Plasmid Maxi Kit (Qiagen, Germany), CPT High-efficiency Transfection Kit (Virotherapy Technologies, Wuhan, China).

Human CDC20, PP1α, and PP1γ were amplified from human testis cDNA (Marathon cDNA; Takara Bio) using Pfu polymerase (Agilent Technologies). CDC20 expression constructs were made using pcDNA5/FRT/TO vectors (Invitrogen) modified to encode the EGFP or FLAG reading frames; PP1 constructs were made using a modified pcDNA5/FRT/TO vector encoding a C-terminal GFPtag. Mutagenesis to introduce phospho-site mutations and resistance to CDC20 small interfering RNA (siRNA) oligo #14 was performed using the QuikChange method (Agilent Technologies). DNA primers were obtained from Invitrogen. For the knockdown of the catalytic subunits of PP1α and PP1γ, siRNA duplexes 5′-UGGAUUGAUUGUACAGAAAUU-3′ and 5′-GCGGUGAAGUUGAGGCUUAUU-3′ targeting the 3′-UTR of PPP1CA and PPP1CC, respectively, were used in Figures 2, 3, and 7 and Supplemental Figure S4. Duplexes targeting the ORFs 5′-CAUCUAUGGUUUCUACGAU-3′ and 5′-GAACGACCGUGGCGUCUCU-3′ for PPP1CA or 5′-GCGGAG­AGUUUGACAAUGC-3′ and 5′-UAGAUAAACUCAACAUCGA-3′ for PPP1CC were used in Figures 5, 6, and 8 and Supplemental Figures S2 and S3. PP1β was depleted using a 3′-UTR duplex 5′-GGGAAGAGCUUUACAGACAUU-3′ targeting PPP1CB. CDC20 was depleted using siRNA duplex #14 5′-CGGAAGACCUGCCGUUACA-3′ (ThermoFisher). PP2A-B55 and PP2A-B56 were targeted with siRNA duplexes that have been described previously (Hayward et al., 2019a (link),c).