Figure 8
Myogenin and MEF-2 bind to the ART1 promoter in myotubes, but not in myoblasts in vivo. The chromatin from C2C12 cells (myoblasts and differentiated myotubes) was cross-linked with formalin. Cells were lysed, the nuclear extracts were prepared and sonicated (four times 15 strokes; output, 70%; duty cycle, 60%; Bandelin Sonopuls GM70, Bandelin, Berlin, Germany). After precipitation with antibodies against myogenin (αMG), MEF-2 (αMEF-2) or with an unspecific IgG isotype antibody (Iso) as a control, the ART1 promoter region containing the E box und the A/T rich element was amplified by PCR from the precipitated DNA. Lanes are input DNA (Input) in dilutions: (1/100) 1:100, (1/10) 1:10 und undiluted (1), no antibody (no), water control (H2O) and DNA ladder (M). The PCR amplification of the desmin gene promoter enhancer [43] which contains a functional E box and a functional MEF-2 binding site served as a positive control whereas the amplification of the proximately part of the HPRT-1 housekeeping gene promoter served as negative control.