Cdk1 cdk2

The main reagents used in this experiment include PF-543 citrate (SPHK1 inhibitor; MedChemExpress, HY-15425A, Monmouth Junction, NJ, USA), PD98059 (MEK1/2 inhibitor; MedChemExpress, HY-12028, Monmouth Junction, NJ, USA), S1P (Cayman chemical, 9002921, Ann Arbor, MI, USA), TY-52156 (S1PR3 antagonist; Cayman chemical, 19119, Ann Arbor, MI, USA), wortmannin (PI3K/Akt antagonist; Cayman chemical, 10010591, Ann Arbor, MI, USA). The specific primary antibodies include against SPHK1 (1:1000, CST, 12071S, Danvers, MA, USA), PBX1 (1:1000, CST, 4342S, Danvers, MA, USA), Phospho-Akt (Ser 473; 1:1000, CST, 4060S, Danvers, MA, USA), Akt (1:1000, CST, 4685S, Danvers, MA, USA), Phospho-p44/42 MAPK (Thr202/Tyr204; 1:1000, CST, 4370S, Danvers, MA, USA), S1PR3 (1:1000, Abcam, ab126622, Cambridge, UK), S1PR1 (1:1000, Abcam, ab23386, Cambridge, UK), CDK4 (1:1000, Santa Cruz Biotechnology, sc-23896, Dallas, TX, USA), CDK2 (1:1000, Santa Cruz Biotechnology, sc-6248, Dallas, TX, USA), CDK1/CDK2 (1:1000, Santa Cruz Biotechnology, sc-53219, Dallas, TX, USA), CyclinD1 (1:1000, Santa Cruz Biotechnology, sc-8396, Dallas, TX, USA), β-actin (1:1000, Santa Cruz Biotechnology, sc-47778, Dallas, TX, USA), goat anti-rabbit and anti-mouse horseradish peroxidases (HRPs; 1:5000, Proteintech, B900610 and B900620, Chicago, IL, USA).

Ventricles were collected at the indicated stages, homogenized in radioimmunoprecipitation assay buffer at 4 °C and quantified by bicinchoninic acid assay. Equal total proteins (20 μg) were resolved in SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Immobilon, Millipore). The membranes were blocked and incubated with primary antibodies at 4 °C overnight before incubated with HRP-conjugated secondary antibodies. Protein bands were detected by enhanced chemiluminescence. Antibodies, including REST (Antibodies-online, ABIN747683, 1:1000), P21 (Abcam, ab109199, 1:500), Cyclin E (Santa Cruz, sc-377100, 1:1000), Cyclin B (Santa Cruz, sc-166210, 1:1000), CDK1/CDK2 (Santa Cruz, sc-53219, 1:1000), and GAPDH (Thermo Fisher, MA5-15738, 1:5000), were used in western blot analysis. Full scans of western blots are shown in Supplementary Figs. 9–14.