TPO controls HIF-1α protein stability. (A) UT-7/TPO cells were starved for 24 hours and then treated with 100 ng/mL hTPO, 5 μM MG-132, or TPO plus MG-132 for the indicated time periods. Nuclear extracts were prepared and HIF-1α expression was analyzed by Western blotting. The position of ubiquitinated forms of HIF-1α is indicated. (B) The film was subjected to densitometric analysis and the proportion of ubiquitinated to total of HIF-1α at 6 hours was calculated. ▪ indicates TPO; ▨, MG-132; and □, both. (C) UT-7/TPO cells were starved for 24 hours and stimulated with 100 ng/mL hTPO for 3 hours. CHX (500 μM) was then added and the cells cultured with or without hTPO for the additional time periods indicated. Nuclear extracts were then prepared and HIF-1α levels were analyzed by Western blotting. (D) The film was subjected to densitometric analysis and HIF-1α levels were plotted. Each point shows an average ± SD of 3 experiments. The half-life of HIF-1α was calculated by Kaleida Graph software (Synergy Software, Reading, PA). ^*P < .05.