Fig. 1.

PSF is a constituent of endogenous PER complexes and is important for clock function. (A) Co-immunoprecipitation of PSF and PER2. Nuclear extracts (CT18) from liver or lung (input) and immunoprecipitates (IP, antibodies at top) from the extracts were probed with antibodies at right. U2AF⁶⁵ and LaminA/C, negative controls; IgG-LC (light chain), positive control. (B) Depletion of PSF increases Per1 transcription. Quantitative RT-PCR assays showing steady-state abundance of indicated pre-mRNAs (normalized to Gapdh mRNA) in mouse fibroblasts after introduction of point-mutant control PSF shRNA (white) or after depletion of PSF by PSF shRNA (black). Shown are mean +/− SEM of triplicate experiments; representative of 3 experiments. (C-E) Short circadian period length caused by depletion of endogenous PSF from fibroblasts. (C) Western blot showing the effect of point-mutant control (Mut) shRNA or PSF shRNA on steady-state level of endogenous PSF. ACTIN, loading control. (D) Circadian oscillations of bioluminescence in synchronized reporter fibroblasts after delivery of control Mut PSF shRNA (yellow) or PSF shRNA (blue). Traces from three independent cultures are shown for each. (E) Circadian periods of fibroblasts in (D) (mean +/− SEM; N = 3 for each condition; t-test, two-tailed).