Determination of serum 4HNE-protein adducts

ELISA [31] and Western blotting [32] are methods for measuring 4HNE-protein adducts. In ELISA assay, a human 4HNE ELISA kit (BG, China) was used. The assay was conducted following the protocol supplied by the manufacturer, using microplate reader (Multiskan MK3, Thermo, USA). A standard curve was used for calculating levels of 4HNE-protein adducts in the samples. In Western blotting, we carefully adjusted samples’ concentration, loaded the same amounts of total serum proteins into each lane, because no protein in serum is suitable for loading control. The positive bands were detected by chemiluminescence, and the signal intensity was analyzed using semi-quantitatively using ImageJ 1.42q.