Figure 4

A. Interaction of FBXL proteins with Cullin1 and Skp1. NIH3T3 cells were transfected with Flag-Fbxl3, Flag-Fbxl21, Flag-Psttm and V5-βTrcp1 expression constructs. Co-immunoprecipitated proteins were analyzed by western blotting with anti-CRY1, anti-CUL1, and anti-SKP1 antibodies. Representative blots from 3 independent experiments are shown.

B and C. FBXL21 interacts with CRY1 (B) and CRY2 (C) in native extracts in a circadian manner. Liver extracts from CT0 to CT24 were immunoprecipitated (IP) with an FBXL21 antibody, and Western blotting was performed with CRY1 and CRY2 antibodies. Lower: quantification of co-immunoprecipitated CRY1 and CRY2 amounts. Data are taken from 2 independent experiments. Error bars show ± range (n = 2). 2-way ANOVA shows statistically significant differences between WT and Psttm for the amount of CRY1 co-precipitated with FBXL21 throughout the circadian cycle (p < 0.0001).

D. Differential effects of FBXL21 and PSTTM on CRY1 stability. 293A cells were co-transfected with indicated constructs. 32 hrs after transfection, cells were treated with 20 μg/ml cycloheximide and incubated for the indicated time before harvest. Western blotting was performed to monitor CRY1 levels using an anti-HA antibody. Lower: Quantification of the effects of FBXL3, FBXL21, PSTTM on CRY1 stability. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). Half life was determined by using nonlinear, one phase exponential decay analysis (the half-life parameter, K, is significantly different in all four conditions: p<0.0001)

E. The Psttm mutation destabilizes FBXL21. 293A cells were transfected with Fbxl21-Flag or Psttm-Flag constructs. Cycloheximide treatment was performed as in D. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). The half-life parameter, K, is significantly different: p=0.0436.

F. Competition between FBXL3 and FBXL21/PSTTM modulates CRY1 degradation. Cycloheximide treatment and CRY1 western blotting were performed as in D. Data for quantification represent 3 independent experiments (Figure S4F). The half-life parameter, K, is significantly different: p<0.0001. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3).

Lower: Competition between Ovtm and Fbxl21/Psttm in CRY1 degradation. Cycloheximide treatment and CRY1 western blotting were performed as in D. Data for quantification represent 3 independent experiments (representative western blots are shown in Figure S4G). The half-life parameter, K, is significantly different: p<0.0001. Error bars show ± SEM (n = 3).

G. In vitro ubiquitination assay. Sf9 cells were infected with the indicated baculovirus constructs. Samples were analyzed by western blotting with an anti-Myc antibody. Upper shows a short exposure image, and the bracket to the right marks polyubiquitinated CRY1. Lower shows a long exposure image, and the bracket to the right marks highly polyubiquitinated CRY1. Results are representative of more than 3 replicates.

H. In vitro ubiquitination was performed as indicated in G. Sf9 cells were infected with the indicated baculovirus. Co-infection of Fbxl baculovirus (Fbxl3+Fbxl21, Fbxl3+Psttm) attenuated the E3 ligase activity of FBXL3. Results are representative of 3 replicates.

I. FBXL3/FBXL21 mediated CRY1 ubiquitination requires Ubc5 as an E2 ligase. Top: Ubc5-mediated robust ubiquitination by FBXL3-SCF complexes and multi ubiquitination by FBXL21/PSTTM SCF complex. Lower: lack of CRY1 ubiquitination in the presence of Ubc13/Uev1A as the E2 ligase. Results are representative of 3 replicates.

J. 293A cells were co-transfected with Cry1-HA, ubiquitin (hUb-HA) and the indicated F-box constructs. Cells were treated with MG132 (10μg/ml) 6 hr before harvest. Whole-cell lysates were analyzed by western blotting with an anti-CRY1 antibody. Results are representative of 3 replicates. See also Figure S4.