(A) Wild-type or Cry1−/−;Cry2−/− (Cry−/−) MEFs stably expressing a control sequence (−) or shRNA targeting Hausp (#1) were subjected to nuclear and cytoplasmic fractionation. Cry1, Hausp, Lamin, and Tubulin were analyzed by IB from fractions harvested at the indicated times following circadian synchronization with dexamethasone (CT, hours). (B) Cry1, Hausp, Lamin and Tubulin were detected by IB in nuclear and cytoplasmic fractions from MEFs treated with vehicle (DMSO, −) or Compound 7 (+). (C) Wild-type MEFs stably expressing control or Hausp-targeting shRNA or Cry−/− MEFs were treated with vehicle (DMSO, −) or MG132 (+) for 6 hr, and lysed in RIPA buffer containing iodoacetamide. 6 mg of RIPA lysates from each condition was subjected to IP with 5 μg of anti-Cry1 antibody. Ubiquitinated Cry1 (Cry1− (Ub)N), Cry1, and Hausp were detected by IB in IPs and whole cell lysates (WCL). (D, F, H) Typical results of continuous monitoring of luciferase activity from MEFs expressing Per2-luciferase fusion protein from a knock-in allele (D and F) or from U2OS cells stably expressing luciferase under the control of the Bmal1 promoter (H) with stable expression of control or either of two shRNA sequences targeting Hausp (D) or treated with Compound 7 and/or AICAR (F and H). (E, G, I) Quantitation of the circadian period of luciferase activity from experiments performed as described in (D, F, H). Data represent the mean ± s.d. for 4–8 samples per condition. **p < 0.01, ***p < 0.001 vs control samples (control shRNA for E or DMSO-treated cells for G and I).