Anti pparα

For the chromatin immunoprecipitation (ChIP) experiments, mice were sacrificed at ZT10 and ZT22. The livers of mice were homogenized in 1× phosphate-buffered saline supplemented with 1% formaldehyde, incubated for 5 min at room temperature, and nuclei and chromatin prepared according to (Schmutz et al., 2010 (link)). Briefly, pure liver nuclei form each mouse were obtained by centrifugation through 2.05 M sucrose cushions and the chromatin in 500 μl of 1% SDS, 20 mM Tris pH 7.4, 150 mM NaCl, and 2 mM EDTA was fragmented by sonication (10×10 s pulses at 50% intensity using a Branson SLPe device equipped with a microtip). After a 10-fold dilution with 1.1% Triton X-100, 20 mM Tris pH 7.4, 150 mM NaCl, and 2 mM EDTA, 200 μl of chromatin were used per reaction. DNA fragments precipitated with anti-REV-ERBα antibody (1:50 dilution; SAB2101632; Sigma-Aldrich), with anti-HNF6 (G-10) (1:30 dilution; sc-376167, Santa Cruz Biotechnology), with anti-PPARα (1:25 dilution; 101710; Cayman Chemical Company, USA) or with anti-PER2 (1:25 dilution; No. 611138; BD Transduction Laboratories) were detected with the reverse transcription PCR primers and probes enlisted in Table S2. Along the samples, 1% of the input was processed and the % of input calculated as precipitated material/(amount of input ×100).

A master mixture of the recombinant proteins (0.1 to 0.5 μg/150 μl) and biotin-labeled DNAs (1 pMol/150 μl) were prepared with binding buffer [10 mM Hepes pH 7.9, 2.5 mM MgCl₂, 50 mM KCl, 150 mM NaCl, 5% glycerol, 1 mM DTT, 0.1% IGEPAL CA-630]. The master mixture was dived into 1.5 ml tubes (150 μl/tube). Then streptavidin-beads (15 μl (50% slurry)/150 μl) and 10- and 30-fold excessive unlabeled competitor DNAs (10 to 30 pMol) were added. After 2 hours of rotation, the recovered proteins were washed 3 times with 1 ml of binding buffer and subjected to Western blot analyses with anti-PPARα (Cayman, #101710) and anti-RXRα (Santa Cruz, ΔN197, sc-774) antibodies. For incubation with WY14,643, 1 μM WY14,643 was added to the binding buffer used for the initial incubation and washing. The signal density of Western blot analyses was quantified with the ImageJ program. The signal density without a competitor was defined as 100.

The following primary antibodies were used for immunoblots: Anti-Hexokinase II (Cell Signaling Technology, 2867), Anti-GLUT1 (Cell Signaling Technology, 12939S), Anti-GLUT4 (Cell Signaling Technology, 2213S), Anti-CPT1β (Abcam, ab134988), Anti-VLCAD (Abcam, ab155138), Anti-Acadm (Santa Cruz, sc365108), Anti-PPARα (Cayman, 101710 and Novus Biologicals, N300-537), Anti-PGC-1α (Millipore, Ab3242), Anti-GAPDH (Cell Signaling Technology, 2118C), Anti-Tubulin (Cell Signaling Technology, 3873S). Secondary antibodies used were Anti-Rabbit IgG, HRP-link (Cell Signal Technology, 7074S) and Anti-Mouse IgG, HRP-link (Cell Signal Technology, 7076S). In all Western blot images shown in figures, individual lanes correspond to individual mice. Molecular weights (MW) are shown. The signal intensity of Western blots was quantified using ImageJ software and then normalized by the corresponding loading control (Tubulin or GAPDH).