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. 2016 Apr 18;7(21):30831–30844. doi: 10.18632/oncotarget.8791

Figure 2. Ionomycin-induced cleavage of nuclear Ku80 but not cytosolic Ku80 in MCF10A cells was attributed to activated calpain.

Figure 2

A. MCF10A cells treated with or without ionomycin at designated concentrations for 2 hours were lysed and prepared for cytoplasm and nucleoplasm separately, followed by Western blotting. Cytosolic Ku70 and Ku80 and nuclear Ku70 were not influenced by ionomycin-induced calcium influx. However, nuclear Ku80 was cleaved in a calcium concentration-dependent manner. The levels of Ku70, Ku80, and cleaved Ku80 proteins in A. were normalized to the β-actin or α-tubulin content for cytoplasm and the histone content for nucleoplasm and depicted as histograms in B. and C. Values represents the mean ± SD of three independent experiments. **p<0.01 vs. the values of ionomycin-untreated MCF10A cells. D. Calpeptin, a calpain inhibitor, blocked cleavage of nuclear Ku80 in a concentration-dependent manner, indicating that translocated m-calpain induced proteolysis of nuclear Ku80. The levels of Ku70, Ku80, and cleaved Ku80 proteins in (D) were normalized using the same method as mentioned above. E, F. Bars represent the mean ± SD of three independent experiments. *p<0.05, **p<0.01 vs. the values of ionomycin-untreated MCF10A cells. †††p<0.001 vs. the values of ionomycin-treated and calpeptin-untreated MCF10A cells.