Figure 3.

BMAL1-dependent induction of Rev-Erbα expression by Ahi1 deficiency. A, schematic representation of wildtype (WT-Luc) and mutant Rev-Erbα promoters (mutant-Luc) that were constructed into PGL3-Basic vector. HEK293 cells were transfected with WT or mutant Rev-Erbα-Luc along with EGFP, EGFP-AHI1 or HA, HA-BMAL1/HA-CLOCK (B/C), respectively. Forty-eight h after transfection, a luciferase reporter assay was performed. ***, p < 0.001; ns, no statistical significance (n = 3). B, N2a cells were transfected with the indicated siRNAs. Seventy-two h after transfection, the cell lysates were subjected to immunoblot analysis. The intensities of BMAL1 and CLOCK relative to GAPDH (bottom) were analyzed. **, p < 0.01 (n = 3). C, the BMAL1 and CLOCK protein levels of Ahi1 KO mouse midbrains and the littermate controls were examined using immunoblot analysis (n = 3). The intensity of BMAL1 relative to GAPDH (bottom) was analyzed. **, p < 0.01 (n = 3). D, N2a cells were transfected with the indicated siRNAs. Seventy-two h after transfection, real-time qPCR was performed. ***, p < 0.001; ns, no statistical significance (n = 3). E, HEK293 cells were transfected with FLAG-RORα along with EGFP or EGFP-AHI1. Forty-eight h after transfection, an immunoprecipitation assay was performed with anti-GFP antibodies. F, HEK293 cells were transfected with FLAG-RORα and EGFP-AHI1 (green). Forty-eight h after transfection, immunofluorescence was performed with anti-FLAG (red). The nuclei were stained with DAPI (blue). G, left, a schematic representation shows the WT and mutant Bmal1 promoter luciferase reporter. Right, HEK293 cells were transfected with WT-Luc or mutant-Luc of Bmal1 promoter along with EGFP, EGFP-AHI1 or FLAG, FLAG-RORα, respectively. Forty-eight h after transfection, a luciferase reporter assay was performed. ***, p < 0.001; ns, no statistical significance (n = 3). Error bars, S.E. IP, immunoprecipitation; IB, immunoblotting.