Wbkls0010

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 proteins were extracted from cell or tissue samples using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors and quantified using a bicinchoninic acid assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 20 μg of denatured proteins in each lane, after which the proteins were transferred to polyvinylidene fluoride membranes. Blocking was performed by incubating the membranes in 5% skimmed milk for 2 h at room temperature. After blocking, the membranes were immersed in diluted primary antibodies (Table 2) overnight at 4°C and washed three times with PBS/Tween 20 (PBST) for 5 min each. Then, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and rinse three times with PBST for 5 min each. An enhanced chemiluminescence reagent (WBKLS0010, Millipore, Burlington, MA) was added for color detection using a Tanon-5200 automatic analyzer (Tanon, Shanghai, China), and the protein bands were visualized and analyzed using Tanon GIS software.

Total protein of the ACC tissues was extracted using RIPA buffer supplemented with a protease inhibitor cocktail. Then, the concentration of protein was determined using the BCA Protein Assay Kit (catalog no. P0010S, Beyotime). Subsequently, 30 μg of protein was loaded per lane. After gel electrophoresis, membrane transfer, and blocking, the membranes were immunoblotted with primary antibodies against 5-HT_1ARs (1:1,000, catalog no. ab85615, Abcam), 5-HT_2ARs (1:100, catalog no. sc-166775, Santa Cruz), 5-HT₇Rs (1:1,000, catalog no. ab128892, Abcam), and GAPDH (1:10,000, catalog no. G9295, Sigma-Aldrich) at 4^°C overnight. After washing, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:2,000, catalog no. ARG65351, Arigo) or horseradish peroxidase-labeled goat anti-mouse IgG antibody (1:2,000, catalog no. ARG65350, Arigo) at room temperature for 2 h. Membranes were revealed using enhanced chemiluminescence reagents (ECL, catalog no. WBKLS0010, Millipore) by a chemiluminescence imaging system (GE Healthcare, United States). The protein expression levels were quantified based on the intensities of the bands with the Image J software and normalized against the GAPDH expression level. Additionally, the membranes reacted with 5-HT receptor antibodies, then stripped, and re-probed with GAPDH antibody in western blot analysis.