Fig. 1. Expression, IGF binding ability, and effects on Igf2 mRNA levels of wtIGFBP-5 and mutIGFBP-5. (A) Western immunoblot to detect the Myc epitope in cell lysates from C2 myoblasts transiently transfected with control vector, wtIGFBP-5 (wtBP5) or mutIGFBP-5 (mutBP5) and transferred to DM for 24, 48 and 72 hours. (B) Ligand blot using biotinylated IGF-I to detect IGFBP-5 in cell lysates from C2 myoblasts transfected as in A and harvested after 48 hours in DM. (C) Northern blot to detect Igf2 mRNA in myoblast lysates after transfection as in A and harvested after 72 hours; (upper panel) the major Igf2 transcript at 3.9 kb and (lower panel) ethidium bromide staining of the 28S and 18S RNA bands as a loading control.