Anti igfbp 2

Fasting blood samples were obtained from all participants and stored for further evaluation, as reported previously.^{15 (link),16 (link)} Circulating IGFBP-2 levels were measured by enzyme-linked immunosorbent assay (ELISA) (catalog no. CSB-E04588h; CUSABIO Corp., Wuhan, China). The intra- and interassay variations in circulating IGFBP-2 measurements were 4.9% and 10.2%, respectively. The IGFBP-2 ELISA results were validated in eight randomly selected serum samples by western blotting. Proteins were extracted using RIPA buffer with added protein and phosphatase inhibitor (Sigma, Darmstadt, Germany), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidine difluoride membranes. Membranes were immunoblotted with anti-IGFBP-2 (catalog no. 3922; Cell Signaling Technology, Danvers, MA, USA) (1:1000) and rabbit monoclonal glyceraldehyde 3-phosphate dehydrogenase (catalog no. ab128915, Abcam Inc., Cambridge, MA, USA) (1:2000) for 12 hours at 4°C. NAFLD was diagnosed according to the guidelines for the diagnosis and treatment of NAFLD issued by the Fatty Liver and Alcoholic Liver Disease Study Group of the Chinese Liver Disease Association.^{17 (link)–19 (link, link)}