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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: J Mol Med (Berl). 2011 Feb 12;89(6):577–593. doi: 10.1007/s00109-011-0732-8

Fig. 6.

Fig. 6

Valosin-containing protein (VCP) regulates HDAC2 and Nrf2 protein levels. a The Beas2B cells were either transfected with control (pSM2) or pSM2-VCPshRNA (48 h) plasmids or treated with PS-341 (1 µM, overnight). The inhibition of VCP protein levels or proteasomal activity leads to upregulation of Nrf2 protein expression. The efficacy of pSM2-VCPshRNA is shown by VCP immunoblotting. b The HEK-293 cells were transiently transfected with VCP-myc plasmid for 48 h. The data indicate that VCP over-expression decreases Nrf2, p-Nrf2, and HDAC2 protein expression levels as compared with Lipofectamine treated controls. The right panel shows the densitometric analysis for (a, b, *p<0.05). c The paraffin-embedded longitudinal human lung sections of control (GOLD 0) and COPD lung tissue at GOLD I–IV levels of emphysema (n=4–5, each group), were immunostained with primary rabbit monoclonal p-Nrf2 and secondary anti-rabbit TR. The data show a significant decrease in p-Nrf2 (red) nuclear localization (co-localization with nuclear Hoechst, blue staining) with increasing severity of COPD lung disease. Statistical analysis (right panel) confirms the significant decrease in p-Nrf2+ nuclei (p<0.001) with progressive lung inflammation and emphysema. d The control and COPD lung tissue sections (n=8–10) immunostained with polyclonal HDAC2 and secondary anti-rabbit-FITC conjugated antibody, present significant decrease in HDAC2 expression and nuclear localization in the lung tissues with moderate or severe emphysema. Hoechst staining of the same area is shown in the bottom panel. Densitometric analysis (right panel) confirms the significant decrease in HDAC2 expression (p<0.001) with progressive lung inflammation and emphysema. Scale=50 µm. e An increase in HDAC2 protein levels is observed in HBE cells upon shRNA-mediated VCP knockdown. The densitometric analysis (mean±SD, p<0.05) of VCP and HDAC2 normalized to β-actin shows a significant increase in HDAC2 expression in VCP shRNA treated cells. The data verify the role of VCP-mediated proteasomal degradation in controlling activated form of Nrf2 (p-Nrf2) and HDAC2 protein levels