Figure 4.

MYOGENIN neutralizing phosphomutant (S160/164A) is resistant to GSK3β repression of transcription activity as well as an increased slower migrating, hyperphosphorylated MYOGENIN band. (a) 4x E-box Luc activity was assessed in C2C12 myoblasts that were transfected with either wild-type MYOGENIN or MYOGENIN (S160/164A) and, co-transfected with HA-GSK3β(S9A) or pcDNA3.1 control plasmid as indicated. HA-GSK3β(S9A) repressed MYOGENIN trans-activation of the 4x E-box promoter region (P<0.001) but had no effect on mutated MYOGENIN (S160/164A) transcriptional activity. (b) Western blot analysis of the same samples revealed a decrease in a slower migrating, hyperphosphorylated band for overexpressed MYOGENIN (S160/164A, lane 2) with respect to overexpressed wild-type MYOGENIN (lane 1). Co-transfected HA-GSK3β(S9A) caused an increase in the slow migrating, hyperphosphorylated MYOGENIN band (lane 3) but not with overexpressed mutated MYOGENIN (S160/164A, lane 4). (c) Independent analysis of E-box Luc activity in C2C12 myoblasts with different combinations of overexpressed MYOGENIN, mutated MYOGENIN (S160/164A), HA-GSK3β(S9A) or pcDNA3.1 control plasmid as indicated. ***P<0.001, **P<0.05, ^#ns