FIGURE 3.

HIF-1α(P402A/P563A) undergoes ubiquitination and proteasome-dependent degradation. A, mutation of Pro⁴⁰² and Pro⁵⁶³ to alanine does not prevent ubiquitination of HIF-1α. HEK 293 cells were cotransfected with pHA-ubiquitin and pFLAG, pFLAG-mHIF-1α, or pFLAG-mHIF-1α(P402A/P562A) (PP-A), and treated with CoCl₂ and the proteasome inhibitor MG132 as indicated (+). Whole cell extracts (inputs) and immunoprecipitated FLAG-tagged proteins (IP) were analyzed by SDS-PAGE and immunoblotting using anti-FLAG (α-FLAG), anti-hemagglutinin (α-HA), or anti-actin (α-Actin) antibodies. B, HIF-1α double proline mutant expressed in HepG2 is stabilized in response to MG132 treatment. HepG2 cells were transfected with 1 μg of HIF-1α and mutant expression plasmids and treated for 12 h with CoCl₂ and MG132 as indicated (+). FLAG-tagged proteins were immunoprecipitated from whole cell extracts (500 μg) and analyzed by SDS-PAGE and immunoblotting using an anti-FLAG antibody (α-FLAG).