HEK293T cells (2 × 107 cells) were reverse-transfected on 15-cm dishes with 32 μg of expression vector by Lipofectamine 2000. After 24 hours, 0 or 8 μM GO289 was applied. After 24 hours, the cells were collected in ice-cold phosphate-buffered saline (PBS) and resuspended in 1.5 ml of incubation buffer [50 mM tris, 50 mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM DTT, cOmplete Protease Inhibitor Cocktail, and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich) (pH 8.0)]. The mixture was supplemented with NP-40 (final 1%) and incubated on ice for 15 min, followed by centrifugation (14,000g) at 4°C for 10 min. The supernatant was used for immunoprecipitation. The following expression vectors were used: C-terminal 3XFlag-tagged PER2, CRY1, and BMAL1 in p3XFLAG-CMV-14 (Sigma-Aldrich); C-terminal 3XFlag-tagged CLOCK in pcDNA3-intron (22); and C-terminal 3XHA-tagged CK2α in pRC-CMV (Addgene #27086).

Before purification, 30 μg of anti-Flag antibody (F1804, Sigma-Aldrich) was cross-linked to 167 μl of Dynabeads Protein G (Invitrogen, 100-09D). Cell lysates (500 μl) were subjected to immunoprecipitation for 1 hour at 4°C. The beads were washed three times with 1 ml of incubation buffer and boiled with SDS sample buffer to elute bound proteins. The proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The band for each protein was cut out and subjected to phosphoproteomics.

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