The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-alpha subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable El protein even at 39.5 degrees C for 12 h, and complete depletion of El protein expression at 39.5 degrees C by siRNA enhanced HIF-1 alpha and P53 protein expression. Further inhibition of El at 39.5 degrees C by siRNA, or El inhibitor Ube1-41 completely blocked HIF-1 alpha degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG HIF-1 alpha plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1 alpha. Additionally, hypoxic HIF-1 alpha degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1 alpha protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. (C) 2016 Elsevier Inc. All rights reserved.