Ecl chemiluminescent kit

Cell proteins were extracted using RIPA lysis buffer and quantified by BCA assay. The proteins were electrophoresed on a polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were coated with a primary antibody (Abcam, UK) overnight at 4 °C. After rinsing with the tris-buffered saline Tween, proteins were cultured with the secondary antibody (Abcam, UK) at room temperature for 2 h. Then, an ECL chemiluminescent kit (Advansta, USA) was used to expose protein bands.

Lysing liver tissues and RAW264.7 cells with RIPA buffer solution for extraction of protein by adopting centrifugal separation. After collection of supernatants, a BCA protein assay kit (Boster, China) calculated protein concentration. Protein lysates which were denatured by SDS underwent SDS-PAGE electrophoresis and were transferred to the 0.22 µm PVDF membrane (Roche, Penzber, Germany). The PVDF membranes were nonspecifically sealed with 5% milk and then washed with TBST (TBS contained 0.1% Tween-20) three times for 10 min each time. Following incubation with the special primary antibodies at 4°C overnight, then the PVDF membranes were flushed three times with TBST buffer. The membranes were then incubated with HRP-labeled goat antibodies against rabbit and mouse IgG (1:5,000, Beijing Zhongshan Biotechnology Co. Ltd., Beijing, China). After the preliminary treatment, the ECL-chemiluminescent kit (ECL, Advansta, United States) was used for detection. The densities of the protein immune response bands were analyzed with image J computer software. The primary antibodies were listed below: Bmal1, IL-10, IL-1β (Affinity Biosciences, Cincinnati, OH, United States), TNF-α, HK2, PFKP, S100A9 (Proteintech, Wuhan, China), ARG-1 (Wanleibio, Shenyang, China).