Ab140592

Cell or tissue samples were lysed using radioimmunoprecipitation assay buffer. Protein content was quantified using a bicinchoninic acid assay kit (PAB180007, Bioswamp), and 20 μg of proteins were loaded onto a 12% gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes and blocked with 5% skim milk at room temperature for 2 h. Then, the membranes were incubated overnight at 4°C with primary antibodies against S1P (rabbit, 1:1,000, ab140592, Abcam), PAR-1 (mouse, 1:1,000, NB1-71779-SS, Novus Biologicals), S1PR1 (rabbit, 1:1,000, NB120-11424, Novus Biologicals), SphK1 (rabbit, 1:1000, ab71700, Abcam), SphK2 (rabbit, 1:1,000, 1-SP030-02, Quartett), IL-1β (rabbit, 1:500, ab200478, Abcam), and GAPDH (rabbit, 1:5,000, 10494-1-AP, Proteintech, Rosemont, IL, USA). The membranes were then washed three times with PBS/Tween 20 for 3 min each and incubated at room temperature for 1 h with goat anti-rabbit IgG (1:10,000, PAB150011, Bioswamp) or goat anti-mouse IgG (1:10,000, PAB150009, Bioswamp) secondary antibodies. After three washes in PBS/Tween 20 for 5 min each, the membranes were incubated with an enhanced chemiluminescence reagent (WBKLS0010, Millipore) in the absence of light and visualized using an automatic analyzer (Tanon-5200, Tanon, Shanghai, China).

Total protein was extracted by suspending the cell pellet in a cell lysis buffer containing 50 mM Tris‐HCl (pH 7.4), 150 mM NaCl, 1% Triton X‐100, 0.1% SDS, and 1 mM EDTA supplemented with protease inhibitor cocktail from Roche (Sigma, St. Louis, MO) (Brown et al., 2011). The BCA kit (Abcam, ab102536) was used to determine the protein concentration. Protein (100 μg) from both control and treated samples was subjected to 4%–20% SDS‐PAGE. The resolved proteins in the gel were transferred to a PVDF membrane electrophoretically. The membrane was separately incubated first with Anti‐SREBP2 antibody (ab30682), Anti‐SCAP antibody (ab190103), Anti‐SP1 antibody (ab227383), Anti‐S1P antibody (ab140592), Anti‐S2P antibody (ab140594), Anti‐GAPDH antibody (ab181602) from Abcam (1:1,000) and then with goat anti‐rabbit secondary antibody (1:10,000) conjugated with horseradish peroxidase followed by ECL detection from Bio‐Rad (Hercules, CA).