FIG. 7.
ActD treatment increases the half-life of HIF-α without a decrease in its nuclear ubiquitination. (A) Cytoplasmic accumulation of endogenous HIF-1α in HeLa cells upon a return to normoxia is inhibited by ActD treatment. HeLa cells were incubated for 8 h in hypoxia in the absence or presence of ActD (10 μg/ml) for the last 2 h. Hypoxic cells were fixed with 1% formaldehyde or fixed 2 or 10 min after reoxygenation and then stained with an anti-HIF-1α antibody. (B) Lower magnification of HIF-1α immunostaining of reoxygenated HeLa cells. Experiments were performed as in panel A. (C) Efficient degradation of endogenous HIF-1α is dependent upon ongoing transcription. HeLa cells were incubated for 8 h in hypoxia in the absence or presence of ActD (10 μg/ml) for the last 2 h. Cells were returned to an oxygenated environment for the indicated times and Western blotting was performed with an anti-HIF-1α. The half-life (1/2t) of endogenous HIF-1α is indicated. (D) Nuclear ubiquitination of HIF-1α is unaffected by ActD treatment. Hypoxic HeLa cells were either not treated or treated with ActD (10 μg/ml) for 2 h and then returned to an oxygenated environment for 2 min. Lysates were incubated with agarose S5a beads, and captured polyubiquitinated proteins were analyzed by SDS-6% PAGE, followed by Western blotting with an anti-HIF-1α antibody. The top panel shows a straight Western blot for HIF-1α content. The experiment was conducted to obtain similar HIF-1α levels in untreated and in ActD-treated cells.