Figure 2

Bag5 is stress-induced and accumulates in the endoplasmic reticulum. Bag5 gene expression is induced upon stress. A. Real time PCR analysis of Bag5 gene in 22Rv.1 cells upon treatment with thapsigargin (TG) or tunicamycin (TN) for the indicated time points. Gene expression values were normalized to Rib36. Bar charts indicate the mean value of three independent experiments ± SD. (* p < 0.05) B. Real time PCR analysis of GRP78 gene in 22Rv.1 cells upon treatment with thapsigargin (TG) or tunicamycin (TN) for 12 h. Gene expression values were normalized to Rib36. Bar charts indicate the mean value of three independent experiments ± SD. (* p < 0.05; ** p < 0.01). C. Bag5 protein expression is induced upon stress. Western blot analysis of 22Rv.1 cell extracts after thapsigargin or tunicamycin treatment for the indicated time points. β-actin was detected as loading control. D. Bag5 accumulates into the ER upon stress. D. Bag5 associates to the ER upon stress. Confocal microscopic analysis of 22Rv.1 cells-paraformaldehyde-fixed and stained with an anti-Bag5 antibody (green channel) or the ER-tracker (red channel) after 12 hours thapsigargin treatment. All images (40X) were acquired with a Leica TCS SPE confocal microscope (Leica Microsystems). The scale bar indicates 25 μm. E. Quantification of the percentage of co-localization performed with the software Imaris CoLoc. Bar charts indicate the average of three fields of three independent experiments ± SD (* p < 0.01). F. ER extraction from 22Rv.1 cell lysates treated with TG for 8 h. Western blot analysis was performed with a Bag5 or a GRP78 specific antibody. Anti-α-tubulin was used for ER fractions quality control (C: cytoplasm; ER: endoplasmic reticulum).