Synergistic Activation of HIV-1 Expression by Deacetylase Inhibitors and Prostratin: Implications for Treatment of Latent Infection
Figure 5
Effect of VPA on prostratin-mediated NF-κB signaling pathway activation.
Nuclear (A) or cytoplasmic (B) extracts were prepared from Jurkat cells mock-treated or treated with TNFα (10 ng/ml), prostratin (5 µM), VPA (2.5 mM) or with prostratin+VPA for different time periods. A. An oligonucleotide corresponding to the HIV-1 LTR NF-κB sites was used as probe in EMSAs with the nuclear extracts. As control for equal loading, the bottom panel shows comparability of the various nuclear extracts assessed by EMSA with an Oct-1 consensus probe. An experiment representative of three independent experiments is shown. B. Cytoplasmic extracts were analyzed by Western blotting using an anti-IκBα antibody (top panel) and an anti-actin antibody (data not shown) as internal control. Levels of IκBα and actin were quantified by chemiluminescence analysis using ChemiDoc XRS System (Biorad) and results are expressed as IκBα amount/actin amount (bottom panel). The ratio obtained with the mock-treated Jurkat cells was arbitrarily assigned a value of 100%. An experiment representative of two independent experiments is shown.