<p>(A) Nuclear localization of NF-κB and p53 in response to ER stress in MCF-7 cells. The cells were treated with 1 μg/ml BFA, and the localization of NF-κB and p53 were determined by using immunofluorescence staining. (B) NF-κB and p53 nuclear localization were observed in a time-dependent manner after 0, 6, 12, and 24 h exposure to BFA in MCF-7 cells. The cells were treated with 1 μg/ml brefeldin A as time indicated, and NF-κB subunits in the cytoplasmic and nuclear fractions were analyzed by Western blotting with antibodies against NF-κB subunits p65, p50, c-Rel, and p53. Topoisomerase II and α-tubulin were used as internal markers for nuclear and cytoplasmic proteins. (C) time-dependent effect of BFA on p53 phosphorylation. MCF-7 cells were treated with 1 μg/ml brefeldin A in 10% FBS–supplemented DMEM as time indicated. The total cell lysates subjected to immunoblotting with antibodies against anti-p53, anti-p-ser<sup>6</sup>-p53, anti-p-ser<sup>15</sup>-p53, anti-p-ser<sup>20</sup>-p53, anti-p-ser<sup>37</sup>-p53, and anti-p-ser<sup>46</sup>-p53. Exposure of MCF-7 cells to 50 J/m2 of UV light served as positive control. (D) ER stress induced transcriptional up-regulation of p53. MCF-7 cells were transfected with reporter vectors encoding firefly luciferase driven by p53 promoter, pp53-TA-Luc plasmid. Transfectants were treated with tunicamycin or Brefeldin A for 24 h. Luciferase activities were normalized to that of cotransfected Renilla luciferase. Columns, mean of three independent experiments; bars, SD (*, <i>P</i><0.05, Student's t test).</p
