Ab233386

When SK-HEP-1 and MCF-7 cells grew to a density of about 80%, we collected them and added cell lysate buffer (Beyotime, Shanghai, China) and isolated the protein. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Boster, Wuhan, China). After SDS–PAGE electrophoresis, proteins were transferred to PVDF membrane. The membrane was incubated with the anti-S1PR1 antibody (Abcam, ab233386) for one night and then an anti-rabbit secondary antibody (Sanjian, Tianjin, China, LK2001) for 2 h. Images were processed with Image J software.

Western blotting experiments were performed as previously described. The primary antibodies used in the western blotting were as follows: RPTOR (1:1000, ab40768; Abcam), YY1 (1:1000, #46395; Cell Signaling Technology [CST]), Ki67 (1:1000, ab16667; Abcam), CD34 (1:1000, ab81289; Abcam), MMP9 (1:1000, ab228402; Abcam), MMP2 (1:1000, ab92536; Abcam), SPHK2 (1:1000, ab264042; Abcam), S1P1 (1:1000, ab233386; Abcam), Stat3 (1:1000, #9139; CST),Phospho-Stat3 (Tyr705) (1:1000, #9145; CST), Phospho-Stat3 (Ser727) (1:1000, #94994; CST), E-cadherin (1:1000, #14472; CST), N-cadherin (1:1000, #13116; CST), Vimentin (1:1000, #5741; CST), Snail (1:1000, #3879; CST), Slug (1:1000, #9585; CST), GAPDH (1:5000, 10494–1-AP; Proteintech), ZO-l (1:1000, #13663; Cell Signaling Technology), Occludin (1:1000, #91131; CST), Claudin 5 (1:1000, ab131259; Abcam). The secondary antibodies used in the western blotting were as follows: HRP- Goat Anti-Rabbit JgG (Immumoway); HRP- Goat Anti-mouse IgG (Immumoway).

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.