Figure 2.

Overexpressed, constitutively active GSK3β (S9A) represses MYOGENIN trans-activation of E-box. (a) C2C12 myoblasts were transfected with 4x E-box Luc reporter and different combinations of HA-GSK3β(S9A) and MYOGENIN or pcDNA3.1 control plasmid as indicated. Overexpressed HA-GSK3β(S9A) repressed MYOGENIN transcriptional activity (P<0.001). (b) GSK3β directly phosphorylates MYOGENIN in vitro: Purified GST-MYOGENIN was incubated in vitro with GST-GSK3β and (γ-³²P) ATP. GST and MBP proteins were used as negative and positive control respectively as indicated. Bands were resolved using SDS-PAGE and visualized by Coomassie Blue staining. Gels were dried and exposed to X-ray film for 21 h after the assay. (c) Calf-intestinal phosphatase (CIP) treatment of immunoprecipitated MYOGENIN that was obtained from 1000 μg of RH30 protein extract. The data shows that CIP treatment causes a loss of a high-molecular weight, phosphorylated form of MYOGENIN. ^#ns, ***P<0.001