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

FIG. 5.

FIG. 5.

Clusterin is a secreted negative regulator of the PI3K-Akt signaling pathway. (A) The High S column-bound proteins from HeLa cell CM (HeLa-CM) or HeLa-PH cells (HeLa-PH-CM) were eluted by the pH gradient. The eluted fractions were analyzed for clusterin (top), and the corresponding fractions were tested for their ability to activate Akt in NB4 cells (bottom). (B) HeLa-PH-CM inhibits the Akt activation elicited by HeLa-CM. HeLa-PH-CM and its fractions shown in panel A were mixed (1:1) with HeLa-CM and tested for their abilities to inhibit HeLa-CM-elicited Akt activation in NB4 cells. (C) The shRNA-mediated knockdown of clusterin in HeLa-PH1 cells is shown. HeLa-PH1 cells were transfected with the empty vector, the control shRNA vector, or two different clusterin shRNA vectors, and then the serum-free CM (24 h) and the whole-cell lysates of each transfectant were analyzed for clusterin expression. (D and E) Each HeLa-PH-CM was mixed (1:1) with HeLa-CM and tested as described in the legend to panel B for the ability to inhibit HeLa-CM-mediated Akt phosphorylation in NB4 or HEK293 cells. The representative Western blotting of at least three independent experiments is shown.