Identification of SUMOylation sites in DEC1.

<p>(A) Putative SUMOylation sites of DEC1. (B) COS-7 cells were transfected with HA-tagged wild-type DEC1 or its mutants K159R, K279R or K159R/K279R (2K/2R) and Myc-tagged SUMO1, and then subjected to western blot with anti-HA antibody. (C) COS-7 cells were transfected with HA-tagged wild-type DEC1 or its mutant K159R/K279R (2K/2R) with or without Flag-tagged SENP1 or SENP1 mutant (mu) and then subjected to western blot with anti-HA antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

Modification of DEC1 by SUMO1, 2 and 3.

<p>(A) COS-7 cells were co-transfected with Myc-tagged SUMO1, 2 or 3 and Flag-tagged DEC1, and then subjected to western blot with anti-Flag antibody. (B) MCF-7 cells were co-transfected with Flag-tagged DEC1 and Myc-tagged SUMO1 and then subjected to immunoprecipitation with anti-Flag antibody followed by western blot with anti-Myc antibody. (C) Untransfected MCF-7 cells were subjected to immunoprecipitation with anti-DEC1 antibody or anti-IgG antibody followed by western blot using anti-SUMO1 antibody. (D) MCF-7cells were cultured in the presence of 10% serum for 24 h. The medium was then replaced with fresh medium containing either 10% or 0.8% serum and further incubated for 12 h, and the cells were finally subjected to western blot with anti-DEC1 antibody.</p

Inhibition of DEC1 ubiquitination by SUMOylation.

<p>(A) MCF-7 cells were transfected with HA-tagged wild-type DEC1 or its mutant 2K/2R and treated with CHX at the indicated time periods. Cell lysate was subjected to western blot with anti-HA antibody. (B) MCF-7 cells were transfected with HA-tagged wild-type DEC1 or 2K/2R together with Myc-tagged ubiquitin. Cell lysate was subjected to IP with anti-HA antibody followed by western blot with anti-Myc antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

Effect of SUMOylation on nucleolar distribution of DEC1 and sensitivity of DEC1 SUMOylation to serum starvation.

<p>(A) MCF-7 cells were transfected with HA-tagged wild-type or mutant (mu) DEC1. Cytosolic and nuclear fractions were subjected to western blot using anti-HA antibody. Fibrillarin and β-actin were measured to monitor the efficiency of nuclear and cytosolic preparations, respectively. The graph shows the intensities of DEC1 bands in the cytosol and nucleus. (B) MCF-7 cells were stained with mouse anti-HA antibody (green) and then counterstained with DAPI (blue) for nucleus detection. MCF-7 cells were transfected with Flag-tagged wild-type DEC1 (C) or HA-tagged DEC1mutant 2K/2R (D) and grown in the presence of 10% FBS before were transferred to a medium containing 0.1% FBS. Cells were collected at the indicated times and subjected to western blot with the corresponding antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

Sensitivity of DEC1 SUMOylation to SENP1.

<p>(A) COS-7 cells were transfected with HA-tagged DEC1 together with 3xE-box-luc reporter and either Flag-tagged SENP1, SENP1 mutant (mu), SENP2 or SENP3. Cells were then collected and subjected to luciferase activity assay. (B) COS-7 cells were transfected as in (A) but with increasing amounts of Flag-SENP1 or SENP1 mutant. (C) COS-7 cells transfected as in (B) were subjected to western blot with anti-Flag or anti-HA antibody. **, <i>p</i><0.01</p

Effect of DEC1 SUMOylation on the repression of CLOCK/BMAL1 activity.

<p>(A) MCF-7 cells were transfected with HA-tagged wild-type DEC1 or its mutant 2K/2R with 3xE-box-luciferase reporter and Flag-tagged CLOCK/BMAL1 and luciferase activity was determined. (B) MCF-7 cells were transfected as in (A) but with increasing concentrations of wild-type DEC1 or 2K/2R and luciferase activity was then determined. (C) Expressions of CLOCK and DEC1 in cells from (B) were detected by western blot with anti-Flag or anti-HA antibody. (D) MCF-7 cells were transfected with HA-tagged wild-type DEC1 or 2K/2R together with Myc-tagged HDAC1. Cell lysate was subjected to IP with anti-HA antibody followed by western blot with anti-Myc antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p