Anti per2

The amount of protein was determined using the BCA protein assay (Thermo Scientific, Rockford, IL, USA), and the same amounts of proteins were normalized for total protein. The protein samples were boiled in RIPA buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and protease inhibitor cocktail; Sigma-Aldrich, St Louis, MO, USA), separated by SDS–polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Non-specific binding was blocked with 5% skim milk in PBS-Tween20, and proteins were probed with anti-EAAC1 (Abcam, Cambridge, MA) at 1:1,000 dilution, anti-PER1 (Abcam) at 1:200, anti-PER2 (Abcam) at 1:1,000, anti-BMAL1 (Abcam) at 1:1,000, anti-CLOCK (Abcam) at 1:1,000 and anti-β-actin (Sigma-Aldrich) at 1:10,000 dilution. After a wash with PBS-Tween20, the horseradish peroxidase-labelled secondary antibodies were probed and detected with the ECL prime HRP detection kit (GE Healthcare, Piscataway, NJ, USA). We performed the quantification of the EAAC1 level using a serial dilution of ZT14 samples as the standard curve.

NK cells from control or chronic shift‐lag mice were transfected with Per1‐siRNA and Per2‐siRNA or negative control siRNA. After 24 hours, the cells were pelleted and lysed in RIPA buffer (Beyotime). Total protein was isolated, and the concentration was estimated using a BCA assay (Beyotime). Equal amounts of protein (50 μg) were separated on 12% SDS‐PAGE gels (Beyotime) and transferred to PVDF membrane (Thermo Fisher). The membranes were blocked with 5% BSA for 2 hours at room temperature and subsequently incubated overnight at 4°C with the following specific antibodies: anti‐Per1 (1:1000, Abcam), anti‐Per2 (1:1000, Abcam), anti‐CLOCK (1:1000, Abcam) and anti‐β‐actin (1:1000, Beyotime). The following day, the membranes were washed and incubated with a secondary antibody (rabbit, 1:1000, Beyotime) for 2 hours at room temperature. Protein bands were visualized using ECL Western blotting substrate on a BioRad ChemiDoc MP system (BioRad, CA).