Nr1d1

Antibody for detecting mouse CRY1 was provided by the laboratory of Aziz Sancar and was described previously (Ye et al., 2011 (link)). Antibodies for detection CLOCK (Bethyl Laboratories), BMAL1 (Bethyl Laboratories), CRY2 (Bethyl Laboratories), NR1D1 (Cell Signaling Technology), and NR1D2 (Santa Cruz Biotechnology) were from commercial sources.
For nuclear fractionation, cells were detached from the plate by trypsinization. After PBS wash, cells were lysed with hypotonic buffer (10 mM HEPES pH7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.3% Igepal-CA630). After centrifugation (400 g for 5 min), the supernatants were collected as cytosol fractions. The nuclear pellets were washed with hypotonic buffer and were lysed with NUN buffer (20 mM HEPES pH7.4, 300 mM NaCl, 1 M urea, and 10% glycerol). After centrifugation (15,000 g for 15 min), the supernatants were collected as the enriched nuclear fractions.
The protein concentrations of lysates were estimated by the QuantiChrom^TM Total Protein Assay Kit (BioAssay System). Equal amounts of total proteins were applied to the Western blot experiments.
The Western blot results were quantified by the Image Lab software (Bio-Rad Laboratories). For estimating the relative levels of CRY1-CLOCK-BMAL1 in the nucleus, CRY1 levels in the nuclear fractions were first normalized to the BMAL1 levels and then compared to the group without 4-OHT treatment.

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.