Rabbit anti nr1d1

Wt and Nrf2-/- Per2:Luc MEFs were cultured to confluence in complete growth medium. Cells were washed once with PBS and changed into serum-free growth medium containing 200 nM dexamethasone and incubated for 2 hr in a standard cell culture incubator for synchronization. Following synchronization, cells were washed once with PBS and complete growth medium was added (t = 0). Cells were harvested at the indicated times post-synchronization as described above. MMH-D3 hepatocytes were cultured, differentiated, and synchronized following the protocol described for the bioluminescence assays. Following synchronization, Recording Medium was added to begin the timecourse. Samples were harvested every 4 hr for 52 hr. For all timecourse assays, 50 μg of total protein extract was resolved on 10% polyacrylamide gels and transferred to PVDF membranes. Membranes were incubated with rabbit anti-PRDX-SO2/3 (Thermo LF-PA0004); rabbit anti-PER2 (Proteintech, 20359); rabbit anti-NRF2 (Santa Cruz, SC-722); and rabbit anti-NR1D1 (Cell Signaling, 13418).

Cells grown to 100% confluence were washed with ice cold PBS and harvested by scraping in cold PBS. Cell pellets were lysed in RIPA buffer [25 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and freshly added Protease Inhibitor Cocktail (Sigma), 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄]. Lysates were cleared by centrifugation at 13,000 g, 10 min, 4°C. Total protein was quantified by Micro BCA Assay (Thermo Fisher). 50 µg of total protein was resolved on 10% SDS polyacrylamide gels. For all western assays, protein was transferred to PVDF membranes. Antibodies used are as follows: rabbit anti-NRF2 (Santa Cruz, SC-722; Proteintech, 16396); rabbit anti-NQO1 (Abcam, AB80588); rabbit anti-NR1D1 (Cell Signaling, 13418). Following incubation with primary antibody, blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hr at room-temperature. Clarity (Bio-Rad, Hercules, CA) substrate was used for chemiluminescent detection using a ChemiDoc (Bio-Rad).