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. 2008 May 5;28(13):4285–4299. doi: 10.1128/MCB.01240-07

FIG. 7.

FIG. 7.

Clusterin inhibits the IGF-1-mediated, but not the EGF-mediated, signaling pathway. (A) Serum-starved NB4 cells were incubated with the untreated [heat(−)] or heat-treated [heat(+)] HeLa-CM for the indicated times, and the activation of the IGF-1 receptor and Akt was analyzed in whole-cell lysates. (B) NB4 cells were preincubated with different amounts of control or IGF-1 receptor-neutralizing antibody in serum-free medium and then incubated with heat-treated HeLa-CM for 30 min before the HeLa-CM was analyzed for phospho-Akt level. (C) The IGF-1-neutralizing antibody blocked the HeLa-CM-elicited Akt activation. The heat-treated HeLa-CM was preincubated with the control or IGF-1-neutralizing antibody, and then the ability to activate Akt was tested in NB4 cells. (D) Secretion of IGF-1 by serum-starved HeLa cells. The levels of IGF-1 in untreated or heat-treated CM collected from HeLa cells or HeLa-PH cells at different time points of serum starvation were measured by ELISA. (E) Functional inhibition of IGF-1 by clusterin. The purified IGF-1 was preincubated with the control (HEK293T-Con-CM) or clusterin-containing CM (HEK293T-Clu-CM) and assayed for the activation of IGF-1 receptor and Akt in NB4 cells. (F) The purified EGF (5 ng/ml) was preincubated with the control or clusterin-containing CM as described in the legend to panel E and assayed for the activation of the EGF receptor, Akt, and Erk in serum-starved HeLa cells at the indicated time points.