Expression of Rad50, enolase, IκB kinase b, and Hsp70 increases upon IGF-1 stimulation of C2C12 cells. A) Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain. The protein highlighted by a yellow square (Rad50, enolase, IκB kinase b, and Hsp70) increase in expression after IGF-1 treatment. PDQuest analysis indicates a quantitative change in the protein based on total quantitation of the entire gel. B) Western blot analysis confirms the sustained increase in protein expression for Rad50, enolase, IκB kinase b, and Hsp70. C) RAD50 accumulates in the cytosolic fraction of IGF-1 stimulated C2C12 cells.