Bmal1

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).

The cells were isolated with lysis buffer, which was further quantified with the BCA method (Elabscience, USA). The 12% SDS‐PAGE was used to separate proteins, followed by transferring them to the PVDF membrane (GE Healthcare Life, USA). After blocking, primary antibodies against Bax (Servicebio, China), P65 (Affinity, USA), p‐P65 (Affinity, USA), Iκ B (Affinity, USA), p‐Iκ B (Affinity, USA), BMAL1 (Affinity, USA), Bcl‐2 (Affinity, USA), and β‐actin (SolelyBio, China) were added and incubated at 4°C for 12 h, followed by the secondary antibody (CST, USA) for 90 min. Last, the level of proteins was quantified by analyzing the bands with the Image J software.