Ab126622

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).

After HUVECs and HepG2 cells were treated with drugs or transfected following the above conditions, the protein was extracted for subsequent experiments. The cells were subsequently lysed with RIPA buffer (Solarbio, Beijing, China). Equivalent amounts of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked in 5% nonfat milk in TBST for 1 h at room temperature and then incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were purchased from Abcam (Abcam, Cambridge, UK) and included antiserine palmitoyltransferase antibody (ab23696), anti-S1P1 antibody (ab233386), anti-S1PR2 antibody (ab220173), and anti-S1P3 antibody (ab126622). The secondary antibodies were incubated at room temperature. The membrane incubates secondary antibodies at room temperature. After the secondary antibody was incubated, the membrane was observed with ECL plus and X-ray film. Finally, the protein concentration was analyzed using ImageJ.