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Figure 2. Clusterin (CLU) gene expression is induced by changes in endoplasmic reticulum stress, caused by thapsigargin (TG) or high dose BAPTA-AM, agents that cause extensive changes in Ca2+ homeostasis. (a) Changes in survival (measured by colony-forming ability; see Materials and Methods section) in mock-treated cells compared with cells that were pre-treated with 3 µM BAPTA-AM. (b) Changes in endogenous 60 kDa pre-secretory clusterin (psCLU) as well as 
40 kDa secretory clusterin (sCLU) levels were monitored in untreated (no treatment, NT) or TG-exposed (2–20 nM) MCF-7 cells. Whole cell protein extracts were prepared at 24 h or 72 h and probed for CLU protein responses by standard Western blot procedure as described previously [11, 13]. Induction of endogenous protein levels was observed by as little as 2 nM TG, which did not cause a significant lethality response (see (a)). Cells harvested 24 h and 72 h after 5 Gy ionising radiation (IR) were used as a positive control. All experiments were performed three or more times, and representative Western blots are shown.
