Indigo program

Total protein was extracted from seedlings grown under LD and harvested at 4h intervals from ZT0 to ZT20 on day 10. Whole protein extract was extracted using a buffer containing 50 mM Na-phosphate pH 7.4, 100 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50 μM MG-132, 2mM Na₃VO₄, 2 mM NaF, and Pierce Protease Inhibitor Tablets, EDTA-free. Approximately 30 μg protein in each sample was run in 12% SDS-PAGE gels, and transferred to Nitrocellulose membranes (Bio-Rad). TCP4-3F6H proteins were detected by using a HRP-conjugated anti-FLAG antibody (A8592, Sigma), whereas histone H3 protein was detected by anti-histone H3 antibody (ab1791, Abcam), followed by a HRP-conjugated goat anti-rabbit antibody (Thermo Fisher Scientific). Immunoreactive proteins were visualized with SuperSignal West Pico Chesmiluminescent Substrate (Thermo Fisher Scientific) and Amersham ECL Select Western Blotting Detection Reagent (GE healthcare). For protein quantification, signals from immunoblottted membranes incubated in chemiluminescent detection reagents were imaged and quantified by a high sensitivity cooled CCD camera system (NightOWL, Berthold) and the IndiGo program (Berthold). Histone H3 was used for normalization of a protein in whole extract.