Effect of WA on relative levels of ubiquitinated protein and proteasomal chymotrypsin-like activity.

<p>A) Cells were treated with either 5 µM withaferin A (WA) for 16 h or with 7 µM A23187, 30 µM MG132 or the appropriate volume of the DMSO vehicle (C) for 24 h at 22°C. Isolated protein was subjected to immunoblot analysis employing a mouse anti-ubiquitin monoclonal antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. The positions of molecular mass standards in kDa are shown in the first lane (M). B) Image J software was used to perform densitometric analysis of the signal intensity for ubiquitinated protein bands of western blot images as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. The data are expressed as a percentage of the lane having maximum density (MG132) while the standard error is represented by vertical error bars. The level of significance of the differences between samples was calculated by one-way ANOVA with a Tukey’s post-test. Significant differences between the control cells and A6 cells treated with 5 µM WA or 30 µM MG132 are indicated as * (<i>p</i><0.05). These data are representative of at least three separate experiments. C) Effect of WA on proteasomal chymotrypsin (CT)-like activity of A6 cells. Cells were treated with WA, A23187 or MG132 as indicated above. A cell-based proteasomal CT-like assay was used to monitor the proteolytic activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050547#s2" target="_blank">Materials and methods</a>. The CT-like activity was measured and expressed as a percentage of the CT-like activity observed in control cells. The level of significance of the differences between samples was calculated by one-way ANOVA with a Tukey’s post-test. Significant differences between control cells and cells treated with WA, A23187 or MG132 are indicated as * (<i>p</i><0.05). These data are representative of three separate experiments.