Figure 6.

PER2 interacts with pGSK3β(Ser9) to enhance active β-catenin mediated Per2 transactivation (see also Figures S7-S9)

(A) Interaction of Per2 with pGSK3β (Ser9) in Per2^wt/GSKß^wt MEFs detected by immunoprecipitation 12 h after LDR followed by immunoblot with anti-pGSK3β(Ser9), or reversely, immunoprecipitation of pGSK3β(Ser9) followed by immunoblot with anti-Per2 antibody (N = negative control without antibody).

(B) Immunoprecipitation of co-transfected V5-Per2 with HA-pGSK3β or HA-pGSK3β S9A-mut 293T cells 12 h following LDR.

(C) Degradation of pGSK3β(Ser9) and active β-catenin measured in Per2^wt and Per2^def BMMNCs 12 h after LDR followed by cycloheximide (30 μg/ml) for indicated times.

(D) Relative expression of pGSK3β(Ser9) and active β-catenin in in Per2^wt and Per2^def BMMNCs 12 h after LDR followed by cycloheximide (30 μg/ml) was quantified with ImageJ and normalized with β-actin levels. Data are represented as mean ± SEM, n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001, ANOVA two-way test was applied.

(E) Western blot of active β-catenin in nucleus and cytosol of Per2^wt/GSK3ß^wt MEFs 8 h after LDR, using histone and β-actin as loading controls respectively for nuclear and cytosol proteins.

(F) Luciferase reporter activity driven by mouse Per2 promoter in Per2^wt/GSK3ß^wt MEFs compared to Per2^wt/GSKß^ko MEFs 12 h after LDR; Per2 luciferase transcription activity was normalized with Renilla activity. Data are represented as mean ± SEM, n = 3, ∗∗p < 0.01, ns p > 0.05, ANOVA two-way test was applied.

(G) Luciferase reporter activity driven by mouse Per2 promoter in Per2^wt/GSK3ß^wt MEFs 12 h after LDR or LDR incubation with 0.1 and 0.3 μm β-catenin inhibitor Calphostin C (Cal, blocking β-catenin transactivation), Per2 luciferase transcription activity was normalized with Renilla activity. Data are represented as mean ± SEM, n = 3, ∗p < 0.05, ∗∗p < 0.01, ANOVA two-way test was applied.

(H) Western blot of PER2 and active β-catenin in Per2^wt/GSK3ß^wt MEFs 12 h after LDR or LDR with Cal (0.1 μm); right, relative expression of PER2 and active β-catenin in Per2^wt/GSK3ß^wt MEFs 12 h after LDR or LDR with Cal (0.1 μm) was quantified with ImageJ and normalized with β-actin levels. Data are represented as mean ± SEM, n = 3, ∗∗p < 0.01, ANOVA two-way test was applied.