Brf1 2

Antibodies against the following were used: Cnot1, Cnot3, Cnot6, Cnot6l, Cnot7, and Cnot8 (mouse monoclonal antibodies; generated by Bio Matrix Research and Research Center for Advanced Science and Technology, The University of Tokyo), BRF1/2 (#2119; Cell Signalling Technology), KSRP (A302-021A; Bethyl Laboratories), α-tubulin (#T9026; Sigma), PER2 (PER21-A, Alpha diagnostic) for mouse tissue, PER2 (STJ115134, St. John’s Laboratory) for HEK293T cells, BMAL1 (A302-616A; Bethyl Laboratories), 4E-BP1 (#9644S, Cell Signalling Technology) and GAPDH (#2118; Cell Signalling Technology).

Four fold of SDS-PAGE sample buffer (200 mM Tris pH 6.8, 8 % SDS, 0.4 % bromophenol blue, 40 % glycerol, 400 mM β-mercaptoethanol) was added to the sample to a final concentration of 1 fold and then heated at 100 °C for 5 min. Proteins were separated on 10 % polyacrylamide gels and transferred onto a 0.45 μm-pore-size polyvinylidene difluoride membrane (Millipore, Billerica, MA) for western blotting. The membrane was incubated for 1 h at room temperature with an antibody against any of the following proteins: BRF1/2, phosphorylated p38 (p-p38) MAPK T180/Y182, p-p44/42 MAPK (all from Cell Signaling), hnRNPC1/C2, MKP-1, ERK1, JNK1 (all from Santa Cruz Biotechnology), total-p38, p-JNK, Flag M2 (all from Sigma-Aldrich), Ttp, Zfp36l1, Zfp36l2, and β-tubulin [26 (link)]. After washing with PBST (PBS containing 0.1 % (v/v) Tween 20) for an appropriate time, the membrane was incubated for 1 h at room temperature with a horseradish peroxidase–conjugated secondary antibody: goat anti-rabbit IgG (KPL, Gaithersburg, ML), goat anti-mouse IgG (KPL), or rabbit anti-goat (Sigma-Aldrich). Western Lightning enhanced chemiluminescence substrate (Perkin Elmer, Norwalk, CT) was used for detection.