Bca method

The SCI areas were lysed using the lysis buffer to obtain total proteins, which were quantified with the bicinchonininc acid (BCA) method (Elabscience, USA). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was used to separate proteins at a 12% concentration and proteins were further transferred to the polyvinylidene fluoride (PVDF) membrane (GE Healthcare Life, USA). After blocking, primary antibodies against Bax (Cat#GB11007, Servicebio, China, 1:1000), Bcl‐2 (Cat#AF6139, Affinity, USA, 1:1000), CD206 (Cat# ab300621, Abcam, UK, 1:2000), CD16 (Cat# ab203883, Abcam, UK, 1:2000), p‐STAT1 (Cat#AF3300, Affinity, USA, 1:1000), TLR4 (Cat#AF7017, Affinity, USA, 1:1000), NF‐κB p65 (#AF5006, Affinity, USA, 1:1000), p‐NF‐κB p65 (Cat#AF2006, Affinity, USA, 1:1000), and β‐actin (Cat#4970, CST USA, 1:10,000) were added to be incubated at 4°C for 12 h. Subsequently, the PVDF membrane was incubated with the secondary antibody (Cat# ab6721, Abcam UK, 1:5000) for 1.5 h, followed by quantifying the level of target proteins by analyzing bands with the Image J software.

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.