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. 2006 Mar 15;395(Pt 1):223–231. doi: 10.1042/BJ20051190

Figure 1. Induction of clusterin gene expression by proteotoxic stress.

Figure 1

(A) Western-blot analysis. After a 16 h incubation with DMSO (control) or MG132 (5 μM), proteins were extracted and assayed for expression of clusterin protein precursor. β-Tubulin was used as a loading control and Hsp70 as a positive control of chaperone induction by MG132. (B) Western blotting showing clusterin protein precursor levels. Induction was analysed by comparison of dilution series (1, 1/2, 1/4 or 1/8) of cellular extracts obtained after a 16 h incubation of cells with DMSO (control) or MG132 (5 μM). (C) Semi-quantitative RT–PCR analysis. U-251 MG cells were treated with DMSO (control), with the proteasome inhibitor MG132 (5 μM) or with L-proline analogue AZC (25 μM). mRNA accumulation for the two internal controls, actin and PO, the positive control Hsp70, and the two members of the sHsp family, clusterin and αB-crystallin, were analysed. The data shown correspond to 28-cycle PCRs. The Figure presented is representative of three independent experiments. (D) Clusterin gene promoter activity. U-251 MG cells were transfected with clusterin (pClu-1297bp) or αB-crystallin (pαBcryst) gene promoter reporter plasmids and the control TK promoter. Cells were treated with increasing amounts of MG132 as indicated for various periods of time (6 or 16 h for clusterin), or 16 h at 5 μM for pαBcryst and TK. Folds of activation for CAT or luciferase activities were expressed as the means±S.E.M. (n=3–10).