Detection of follistatin and myostatin mRNA and protein in ovine primary myoblast cells. A, determination of follistatin mRNA and myostatin. B, mRNA expression by RT-PCR in primary muscle myoblasts and ovine fetal skeletal muscle, as a control. M is 100 bp DNA ladder marker. C, detection of ovine follistatin protein in primary myoblasts via western blot analysis. 15 μg of total protein was resolved by 12% SDS-PAGE. Polyclonal anti-follistatin (Santa Cruz) was the primary antibody used in the western blot. D, detection of myostatin protein in ovine primary myoblast cells and ovine fetal skeletal muscle extracts by western blot analysis. Twenty micrograms of total protein from ovine fetal muscle and 60-day-old ovine primary myoblasts was resolved by 10% SDS-PAGE, and myostatin protein was detected with rabbit anti-myostatin antibodies. Precursor and processed forms of myostatin are indicated. Molecular masses of the western positive bands are indicated. E, Densitometry analysis of protein expression for western blots (myostatin and follistatin) was normalized to GAPDH expression in ovine primary myoblast cells over a time-course of 8 days.