Ab3443

Immunoprecipitation assays were performed as described previously^{22 (link),23 (link)} with slight modification. For ChIP assays, cross-linked chromatin was immunoprecipitated with 5 μg of antibody (anti-HNF-1α, Cell Signaling Technology, #89670; anti-PER1, Abcam, ab3443), or negative control rabbit IgG (Beyotime, A7016) at 4 °C overnight. Immunoprecipitated DNA was then used as a template for PCR. All primer sequences used for ChIP-PCRs were listed in Table S1.
For co-immunoprecipitations, liver tissues were homogenized and lysed with a Non-denaturing lysis buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), and 1% NP-40 with protease inhibitor cocktail (Boster Biological Technology). To prepare immunoprecipitates, we incubated lysates with antibody (anti-HNF-1α, Cell Signaling Technology, #89670; anti-PER1, Abcam, ab3443) overnight at 4 °C, and then incubated with Protein A-Sepharose 4B (Invitrogen). Immunoprecipitates were washed five times with wash buffer containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.2 mM sodium orthovanadate with protease inhibitor cocktail, boiled in SDS–PAGE loading buffer. Proteins were analyzed by western blotting as described above.

Mice were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS) at the indicated time of day (TOD), and the isolated brains were postfixed in the same fixative for 24 h. The brains were then cryoprotected in 30% sucrose and sectioned at 20-µm thickness on a cryostat. The SCN-containing brain sections were blocked by incubation with PBS containing 10% horse serum and 0.3% Triton X-100 for 30 min. Primary antibodies against BMAL1 (ab3350, Abcam, Cambridge, UK), PER1 (ab3443, Abcam) and PER2 (PER21-A, Alpha Diagnostic Int’l, San Antonio, TX, USA) were applied for 1 h. After washing six times with PBS, the sections were incubated with a Cy3-labeled anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratory, West Grove, PA, USA) for 0.5 h. Subsequently, the sections were washed six times with PBS, mounted with 4′,6′diamidino-2-phenylindole (DAPI)-containing medium (Sigma-Aldrich, St. Louis, MO, USA) and observed under a confocal microscope (LSM510; Zeiss, Goettingen, Germany). BMAL1-, PER1- and PER2-immunoreactivity (ir) was quantitatively analyzed using the National Institutes of Health (NIH) program ImageJ (available at https://imagej.nih.gov/ij/) and then normalized against the DAPI signal intensity.