Figure 5. Hypoxia Induces Proteolytic Cleavage of p75^NTR by γ-secretase to Control Siah2 Abundance and HIF-1α Stabilization.

(A) Western blot for p75^NTR in p75^NTR−/− MEFs electroporated with p75FL and subjected to 5 hours of normoxia or hypoxia (1% O₂). Cells were treated with 1 μM of compound E and 20 μM of TAPI for 14 and 2 hours before hypoxia. Based on the predicted molecular weight, the arrows indicate the full length receptor (~75 kDa, FL), the carboxy-terminal fragment (~25 kDa, CTF) or the intracellular domain (~20 kDa, ICD). β-Actin loading controls were performed on the same membrane. Representative image from n=3 independent experiments is shown.

(B) Western blot for p75^NTR in WT MEFs subjected to 5 hours of normoxia or hypoxia (1% O₂). Cells were treated with 1 μM of compound E and 20 μM of TAPI for 14 and 2 hours before hypoxia. MG132 was added 4 hours before hypoxia. Representative image from n=3 independent experiments is shown.

(C) Western blot for HIF-1α expression in WT MEFs in normoxia and during 2, 4 and 6 hours of hypoxia (1% O₂). Cells were pretreated with 1 μM of compound E 14 and 2 hours before hypoxia. β-actin loading controls were performed on the same membrane. The ratio HIF1-α/β-actin is normalized to the non-treated WT cells after 6 hours of hypoxia.

(D) Western blot for HIF-1α and Siah2 in WT MEFs treated with 1 μM of compound E for 14 hours before 5 hours of hypoxia (1% O₂). β-Actin loading controls were performed on the same membrane. Representative images from n=3 independent experiments are shown.

(E) Western blot for HIF-1α and p75^NTR in p75^NTR−/− MEFs transfected with p75FL, p75ICD and p75-FasTM and subjected to 5 hours of normoxia of hypoxia (1% O₂). β-Actin loading controls were performed on the same membrane. Representative image from n=3 independent experiments is shown.

(F) Co-immunoprecipitation of Myc-Siah2 with HA-ubiquitin (HA-Ub) in HEK293T cells transfected with p75FL, p75FasTM or pCDNA3 subjected to normoxia or hypoxia (1% O₂) for 5 hours. Lysates were immunoprecipitated with anti-Myc antibody, and western blots were developed with anti-HA, and anti-Siah2 antibodies. Western blot of total cell lysates developed with anti-p75^NTR and anti-Siah2 antibodies is shown. The Ubiquitin/Siah2 ratio and the Siah2/β-actin ratio determined by densitometry are shown for the IP fraction and total cell lysate respectively. Representative images from n=2 independent experiments are shown. See also Figure S3.