P jun

For total protein extraction, cells were disrupted in RIPA (Applygen, Beijing, China) lysis buffer containing a Protease and Phosphatase Inhibitor Cocktail (Beyotime, Shanghai, China) at 4 °C for 30 min. The cell lysates were centrifuged at 12,000 × g for 10 min at 4 °C and the supernatants were collected. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to quantify the protein concentrations of each sample. Protein aliquots were separated on 10% SDS-PAGE and proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Billerica, USA). The blots were then blocked in 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies at the recommended dilutions with gentle agitation overnight at 4 °C. The primary antibodies included SPHK1, ATF3, p-NF-κB-p65, NF-κB-p65, p-STAT3, STAT3, p-p38-MAPPK, p38-MAPPK, p-ERK1/2, ERK1/2, p-JNK, JNK, p-JUN and JUN (Cell Signaling Technology, Danvers, USA), PTX3 and GAPDH (Proteintech, Rosemont, USA). After washing, the blots were incubated with the corresponding HRP-linked secondary antibody (Cell Signaling, Danvers, USA). Stained bands were visualized with a Tanon 5200 Automatic Imaging System (Tanon, Shanghai, China) using a hypersensitive ECL solution (Applygen, Beijing, China).