Af6005

The liver tissues were prepared as serial sections as described above and treated with citric acid antigen repair buffer (pH =6.0). The liver sections were the incubated with anti-CHOP antibodies (Affinity, AF5280), anti-SPHK1 antibodies (Affinity, AF6005), and anti-S1P antibodies (Affinity, DF4159) overnight at 4 ℃. After rewarming for 1 hour at 37 ℃, the tissue sections were incubated with the secondary antibodies conjugated with horseradish peroxidase (HRP; ab150083, 1:1,000; Abcam, USA) at 37 ℃ for 1 hour. The Nikon Eclipse TS100 microscope was used to obtain images.

Western blot analyses were performed to examine the protein expression in BRL-3A cells or liver tissues. Total protein from BRL-3A cells or liver tissues were extracted using a lysis buffer (P0013B, Beyotime). The protein concentrations were determined using the BCA Protein Assay Kit (P0010, Beyotime). A total of 40 µg proteins per sample was separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore). The membranes were incubated in 5% non-fat milk solved in Tris-buffered saline Tween (TBST) for 1 hour at room temperature, followed by incubation with anti-CHOP antibodies (Affinity, AF5280), anti-SPHK1 antibodies (Affinity, AF6005), anti-S1P antibodies (Affinity, DF4159), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (ab9485; Abcam) overnight at 4 ℃. Membranes were then incubated with diluted HRP conjugated secondary antibody (1:5,000, ab6721; Abcam) for 1 hour at 37 ℃. An enhanced chemiluminescence (ECL) enhanced chemiluminescence substrate was used to visualize the protein bands using an odyssey system (Li-COR, USA). Signal intensity was quantified using ImageJ software (National Institutes of Health) with background subtraction.