Fig. 7. Isolation and analysis of the GFP-myosin expressed in C2C12 myotubes. (A) SDS-PAGE of myosin isolated from uninfected C2C12 myotubes (lane 1), myotubes infected with WT AdGFPMHC and expressing GFP-myosin (lane 2), and purified GFP-myosin sample after ion-exchange chromatography (lane 3). The myosin heavy and light chains are resolved in this 12.5% acrylamide gel. The GFP-myosin heavy chain migrates just above the C2C12 myosin heavy chain in lane 2. (B) A 6% acrylamide gel clearly resolves the GFP-myosin heavy chain from the C2C12 myosin heavy chain: lane 1-Purified adult chicken pectoralis muscle myosin; lane 2-myosin isolated from infected C2C12 cells; lane 3 - Myosin from uninfected C2C12 myotubes. A western blot of the same samples developed with mAb 12C5.3 that selectively reacts with the chicken muscle myosin and labels the 250 kDa GFP-myosin heavy chain, but does not detect the C2C12 myosin. (C) SDS-PAGE analysis of myosin isolated from C2C12 myotubes of uninfected cells (Lane 1), and cells expressing WT GFP-myosin (Lane 2), R403Q (Lane 3), R453C (Lane 4) and G584R (Lane 5). These cells were harvested 96 hours post-infection. Densitometry of the stained gel reveals that the GFP-myosin amounts to 25-40% of the total myosin from the infected myotubes. (D) Immunoprecipitation of the GFP-myosin under native conditions with two anti-myosin mAbs (12C5.3 and 10F12.3) that uniquely recognize the chimeric GFP-myosin reveals that the chimeric protein is predominantly a homodimer.

Return to article