Tissues were snap-frozen in liquid nitrogen immediately after dissection and stored at −80°C until used. For total protein extraction, tissues were homogenized by Bead Ruptor24e (OMNI) with stainless steel beads, in ice-cold RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8, and 1 mM dithiothreitol), supplemented with: protease inhibitors cocktail (1 mM N-(a-aminoethyl) benzene-sulfonyl fluoride, 40 mM bestatin, 15 mM E65, 20 mM leupeptin, and 15 mM pepstatin) (Sigma), PMSF (1:200), Vannadate (1:500), NaF (1:1000). The extracts were centrifuged at 16,200 x g for 15 min at 4°C.
Protein concentration was determined by the BCA assay (Thermofisher), and equalized between samples. Then, samples were heated at 95°C for 5 min in Laemmli sample buffer and analyzed by SDS-PAGE and immunoblot. Antibodies that were used: Rabbit anti-ARNTL15 (link), anti-PER215 (link), anti-p-NR1D1 (Cell Signaling Technology), and Mouse anti-U2AF (Sigma). All antibodies were used diluted 1:1000 in suitable buffer (PBS-T supplemented with 5% BSA, 0.02% sodium azid, and phenol red).
Protein concentration was determined by the BCA assay (Thermofisher), and equalized between samples. Then, samples were heated at 95°C for 5 min in Laemmli sample buffer and analyzed by SDS-PAGE and immunoblot. Antibodies that were used: Rabbit anti-ARNTL15 (link), anti-PER215 (link), anti-p-NR1D1 (Cell Signaling Technology), and Mouse anti-U2AF (Sigma). All antibodies were used diluted 1:1000 in suitable buffer (PBS-T supplemented with 5% BSA, 0.02% sodium azid, and phenol red).