Supplementary Figure 5: Specific regulation of Period 1 and Period 2 mRNA translation by eIF4E phosphorylation

(a) and (b) Puromycin incorporation assay to measure global protein synthesis. For this experiment, animals were injected with puromycin (20 mg/kg, i.p.) and sacrificed 45 min after injection. Brains were harvested and processed for immunolabeling. (a) Representative images of puromycin labeling. Scale bar: 100 μm. (b) Quantitation of the labeling intensity reveals no significant difference in global protein synthesis rates between WT and KI SCN. (c) and (d) Effects of cercosporamide on Per expression. (c) Western blots indicating that the MNK inhibitor cercosporamide did not reduce PER protein levels in KI MEFs. Full-length blots/gels are presented in Supplementary Figure 6. (d) Cercosporamide does not inhibit translation of Per1 and Per2 5’UTR mRNA reporters. (e) Polysome profiles from forebrain lysates. From top to bottom: on the left are qRT-PCR results of levels of Clock, Bmal1, Cry1 and Cry2 mRNA extracted from the polysome fractions. qRT-PCR results on total mRNA extracted from the brain lysates are shown to the right. Note that no significant shift was detected in distribution profiles of Clock, Bmal1, Cry1 and Cry2 mRNAs. No difference was found in Clock, Bmal1, Cry1 and Cry2 mRNA levels between WT and KI mice (n=3, P>0.05 by Student’s t-test).