FIG. 8.
Clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor. (A) The time course analysis of IGF-1-induced Akt activation in the presence or absence of clusterin. The purified IGF-1 (2 ng/ml) was preincubated with the control (Con) or clusterin-containing (Clu) CM and treated in NB4 cells for the indicated times and assayed for the IGF-1 signaling components. (B) The purified IGF-1 was treated as described in the legend to panel A, and the amount was determined by ELISA. (C) The HEK293T-CM collected after transfection with the control or the C-terminal His6-tagged human or zebrafish (zfish) clusterin vector was preincubated with purified IGF-1 and pulled down with Ni-NTA agarose beads and analyzed for IGF-1. (D) Serum-starved HEK293 cells were stimulated; purified IGF-1 was treated as described in the legend to panel A and then immunoprecipitated with IGF-1b receptor. The immune complex was analyzed for IGF-1 by ELISA.