Pcdna5 frt to vector

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).