Fig. 5

IGFBP-3 is required for Capan-1 cells to induce muscle wasting. a IGFBP-3 protein levels in normal C2C12 myoblasts, normal Capan-1 cells, and Capan-1 cells infected with si-GFP or si-IGFBP-3 lentivirus for 48 h were indicated by immunoblot (Up). IGFBP-3 levels in normal growth medium (Con) and conditioned medium of Capan-1 cells infected with si-GFP (CM) or si-IGFBP-3 (CM-I) lentivirus were indicated by immunoblot (Down, non-specific band of IGFBP-3 antibody were used as loading control). b IGF signaling, which is indicated with pAKT levels, in C2C12 myoblasts that were treated with normal DMEM growth medium (Con) or conditioned growth medium from si-GFP (CM) or si-IGFBP3 (CM-I) Capan-1 cells for 24 h (UP); or with Con, CM, or CM that was pre-treated with 5 μg/mL or 10 μg/mL IGFBP-3 antibody for 30 min (CM + Ab) for 24 h. c Myoblasts were seeded at density of 20,000 cells/well and were grown with Con, CM, CM-I, or CM + Ab (10 μg/mL IGFBP-3 antibody) growth medium for 96 h. d Myotubes were differentiated in Con, CM, CM-I, or CM + Ab (10 μg/mL IGFBP-3 antibody) differentiation medium for 96 h. e-g C2C12 myotubes were normally differentiated for 96 h and then incubated with Con, CM, CM-I, and CM + Ab (10 μg/mL IGFBP-3 antibody) differentiation medium for 48 h. The protein degradation (f) and ubiquitinated protein levels (g) were measured. Data are presented as means ± SEM. * p < 0.05