Effect of proteasomal inhibitors on Bag3-mediated downregulation of Large T antigen expression.

<p>A. Effect of Bag3 on transcription of JCV promoters. Primary culture of human fetal glial cells was transfected with CAT reporter genes under the control of JCV early or late gene promoters along with a plasmid expressing Bag3 (pCDNA6-Bag3), or a control empty vector (pCDNA6). Relative promoter activity was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045000#s4" target="_blank">Materials and Methods</a>. B. Protein extracts were prepared from BSB8 cells transfected with plasmid vector (control) or expression plasmid encoding Bag3. Large T-Ag was immunoprecipitated with a monoclonal antibody, separated on SDS-PAGE gel, and processed for Western blotting using anti-Ubiquitin antibody. Normal mouse serum (NMS) was used as negative control for immunoprecipitations. C. BSB8 cells were transfected with an expression plasmid encoding Bag3, and treated with MG-115 or DMSO (control). Whole cell extracts were prepared at 48 hour post-transfection, and Western blot analysis was performed for the detection of Large T, and Bag3 proteins. Western blot analysis of the same extracts with anti-Grb2 antibody was used as loading control. D. Relative band intensity of T-Ag bands from Panel B is shown as a bar graph.