Figure 5.

Ankrd2 regulates the transcription of Gsk3β. (a) Schematic representation of putative NF-κB-binding elements within the Gsk3β promoter identified using Genomatix and Patch software. The white bars depict the regions of the Gsk3β promoter amplified by primer sets covering the putative NF-κB response elements. The Gsk3β luciferase reporter construct is represented on the bottom. (b) Using anti-Ankrd2 antibody, ChIP was performed on nuclear extracts from proliferating and differentiating C2C12 myoblasts. qRT-PCR was performed on input and immunoprecipitated DNA with the Gsk3β promoter-specific primers shown in (a). The bars represent the fold enrichment of Ankrd2 occupancy of the Gsk3β promoter relative to that of control IgG samples. (c) ChIP was performed using anti-p50 and anti-p65 antibodies followed by qRT-PCR analysis as described in (b). (d) Luciferase activity assay following cotransfection of C2C12 cells with a luciferase reporter plasmid encoding the identified Ankrd2 and p50-binding region within the Gsk3β promoter (765 bp, (a)) and WT or S99A mutant Ankrd2. pGL4.74 was used for normalization of transfection efficiency. MB, proliferating myoblasts, MT, differentiating myotubes at 1, 5, and 6 days (d) after induction. All data are represented as mean±S.D. (n=3). *P<0.05; **P<0.01