Hypoxia-inducible Factor-1 Stabilization in Nonhypoxic Conditions: Role of Oxidation and Intracellular Ascorbate Depletion

Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Thrombin modifies HIF-1α protein ubiquitination. Quiescent VSMC were maintained under control conditions or in the presence of thrombin (5 units/mL) for 4 hours. Cytoplasmic extracts were prepared and incubated with in vitro translated and ³⁵S-radiolabeled HIF-1α for in vitro ubiquitination (see Methods). Samples were then resolved by SDS-PAGE (6%). Gels were dried and exposed for ³⁵S autoradiography.
• Figure S2 - Impact of intracellular ascorbate on HIF-1α mRNA levels. Quiescent VSMC were pretreated or not with ascorbate (5µM) for 15 minutes and maintained under control conditions or in the presence of Ang II (100 nM) for 4 hours. Total RNA was extracted and resolved in formaldehyde/agarose gels. Northern blots were performed using a specific radiolabeled cDNA probe comprising the first 900-bp coding sequence of the human HIF-1α gene. An 18S RNA probe was used as a control for gel loading.