Bmal1

SCN tissues from rats and NIH3T3 cells were lysed in RIPA buffer (Huaxingbio, Beijing, China) containing protease inhibitor (Aoqing Biotech, Beijing, China) and phosphatase inhibitor cocktails (Aoqing Biotech, Beijing, China) on ice for 30 min. Each protein was isolated by centrifugation at 12,000× g at 4°C for 20 min. The 30 μg protein was then subjected to 12.5% or 7.5% SDS-PAGE and transferred to a 0.2 μm PVDF membrane. The membranes were blocked with 5% skimmed milk and incubated overnight at 4 °C with specific primary antibodies. The primary antibodies used were as follows: NR1D1 (1:1000; Cell Signaling Technology, 13418, Danvers, MA, USA), GAPDH (1:1000; Proteintech, 60004-1-Ig, Wuhan, China), BMAL1 (1:1000; Cell Signaling Technology, 14020, Danvers, MA, USA), LC3 (1:1000; Cell Signaling Technology, 83506, Danvers, MA, USA), TOM20 (1:1000; Proteintech; 11802-1-AP, Wuhan, China), ATG5 (1:1000; Cell Signaling Technology, 12994, Danvers, MA, USA), and p62 (1:1000; Cell Signaling Technology, 23214, Danvers, MA, USA). For secondary antibodies, HRP-conjugated affinipure goat anti-mouse or anti-rabbit IgG(H + L) (1:10,000; Proteintech; SA00001-1 and SA00001-2, Wuhan, China) were used.

Denatured nuclear extractions or proteins were loaded onto polyacrylamide gels, and after electrophoresis, proteins were transferred onto PVDF membranes (Millipore), then blocked in 5% non-fat milk for 1 h at 25 °C. Incubation of primary antibodies was done overnight at 4 °C in 5% non-fat milk on TBS with 0.1% Tween-20. Primary antibodies were as follows: Med1 (CRSP1/TRAP220) (Bethyl, Cat.: A300-793A, 1:1000), Bmal1 (Cell Signaling Technology, Cat.: 14020 S, 1:1000), Rora (Proteintech, Cat.: 10616-1-AP, 1000), Pparδ (Proteintech, Cat.: 60193-1-Ig, Clone No.: 1B10E1, 1:1000), Tmem173 (Proteintech, Cat.: 19851-1-AP, 1:1000), Nfκb2 (Proteintech, Cat.: 10409-2-AP, 1:1000). The uncropped scans of blots of Bmal1, Rora, Pparδ, Med1, Tmem173, and Nfκb2 were represented in Supplementary Fig. 12.

Hippocampal tissue samples were homogenized in a lysis buffer (Kurabo, Osaka, Japan). The homogenates were centrifuged, and the supernatants were obtained. Western blotting was performed as previously described [24 (link)]. The membranes were incubated at room temperature for 1 h with primary antibodies against brain and muscle arnt-like 1 (Bmal1) (1:1000; Cell Signaling Technology, Danvers, MA, USA), Cryptochrome 1 (Cry1) (1:1000; Proteintech Group, Rosemont, IL, USA), Cry2 (1:1000; Proteintech Group, Rosemont, IL, USA), ionized calcium-binding adapter protein 1 (Iba1; marker of microglia (1:1000; Wako, Osaka, Japan), CC-Chemokine receptor 7 (CCR7) (1:1000; Abcam; Cambridge, MA, USA), and β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA) as loading controls. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were captured using Multi Gauge Software ver. 3.0 (Fujifilm, Greenwood, SC, USA).