Withaferin A Induces Proteasome Inhibition, Endoplasmic Reticulum Stress, the Heat Shock Response and Acquisition of Thermotolerance (original) (raw)
Figure 3
Effect of WA on BiP, GRP94, HSP30 and HSP70 accumulation.
A) Cells were treated with 5 µM WA for 16 h, 7 µM A23187, 30 µM MG132 or the appropriate volume of the DMSO vehicle (C) for 24 h at 22°C. Cells were harvested and total protein was isolated. Forty µg of the different protein samples were analyzed by Western blot analysis using anti-BiP, anti-GRP94, anti-HSP30, anti-HSP70, anti-AKT or anti-actin antibodies as described in Material and methods. Image J software was used to perform densitometric analysis of the signal intensity for BiP, GRP94, AKT, HSP30 and HSP70 protein bands of western blot images as described in Materials and methods. The data are expressed for each treatment as a ratio to control levels (for BiP, GRP94 and AKT accumulation) or as percentage of the maximum signal (30 µM MG132 for HSP30 and HSP70 accumulation). 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 treated cells are indicated as * (p<0.05) or Δ (p<0.10). These data are representative of three separate experiments.