Western Blot Analysis of Serum Proteins

The expression of the selected proteins in serum samples was verified by western blotting (WB), as previously reported [11 (link), 19 (link), 27 (link)]. Briefly, 30 µg of protein from each sample were separated by SDS-PAGE and transferred onto NC membranes (Millipore, Bradford, MA, USA). Then, the membranes were blocked for 2 h in PBST buffer containing 5% bovine serum albumin (BSA) and probed with IL1RL1 and THBS1 primary antibodies (1:1000 dilution, Santa Cruz Biotechnology, USA) and a β-actin antibody (1:5000 dilution, TransGen Biotech, China) at 4 °C overnight. After washing 3 times with PBST buffer for 10 min, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5000 dilution, TransGen Biotech, China) for 1 h at room temperature. Following another wash in TBST buffer, the protein expression levels were detected by enhanced chemiluminescence and visualized by autoradiography. Quantification of the western blot band intensity was performed using ImageJ 1.45 software (NIH) according to the manufacturer’s instructions.