A Novel Function of Novobiocin: Disrupting the Interaction of HIF 1α and p300/CBP through Direct Binding to the HIF1α C-Terminal Activation Domain
Figure 4
Novobiocin directly binds to HIF1α CTAD.
A. Novobiocin-sepharose pulled down HIF1α CTAD. Immobilized novobiocin-sepharose was prepared as described under §Experimental Procedures. GST fusion HIF1α CTAD (∼5 µg) was mixed with novobiocin-sepharose or novobiocin-free sepharose in a buffer containing 40 mM Tris-HCl (pH 7.4), 1% NP-40, 2 mM EDTA, 100 mM NaCl, and 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Sigma). Protein mixtures were incubated at 4°C for 2 hours, and the beads were washed with ice-cold buffer. Bound proteins were eluted by boiling in 4×SDS loading buffer and were separated by SDS-PAGE, followed by Western blotting with anti-GST antibody. B. Novobiocin specifically binds to HIF1α CTAD. Protein mixtures of GST-HIF1α CTAD, novobiocin-sepharose and free novobiocin from 0.25 to 8 mM were incubated at 4°C for 2 hours, and the beads were washed with the same ice-cold buffer. Bound proteins were confirmed by Western blotting with anti-GST antibody (upper panel). Quantified proteins are shown in the bar graph (lower panel). Percentages above each bar represents bound HIF1α 776 protein compared to lane 2. C. HIF1α CTAD and p300 CH1 cannot compete for binding to novobiocin. Pre-equiliblited novobiocin-sepharose (∼20 µl) was mixed with ∼5 µg GST-HIF1α CTAD and Flag-p300 CH1 (5, 10, and 15 µg) in combinations indicated in the figure. Bound proteins were detected by Western blotting with indicated antibodies.