Figure 1. Stabilization of HIF-1α Is Impaired in p75^NTR−/− Cells during Hypoxia.

(A) Western blot for p75^NTR in MEFs and CGNs. β-Actin loading controls were performed on the same membrane. Representative western blot from n=2 independent experiments is shown.

(B) Western blot for HIF-1α during normoxia and after 5 hours of hypoxia (1% O₂) in WT and p75^NTR−/− MEFs and CGNs. β-Actin loading controls were performed on the same membrane. Values are mean ± SEM of the HIF1-α/β-actin ratio normalized to hypoxic WT cells as determined by densitometry (MEFs, n=4; CGNs, n=3 independent experiments, ***P< 0.001 and **P<0.01 by one-way ANOVA). N, normoxia; H, hypoxia.

(C) Kinetics of HIF-1α stabilization in hypoxia (1% O₂) in WT and p75^NTR−/− MEFs by western blot analysis. β-Actin loading controls were performed on the same membrane. The pharmacologic inhibitor DFO that stabilizes HIF-1α was used as a positive control. HIF1-α/β-actin ratio determined by densitometry is shown. Values are mean ± SEM of the HIF1-α/β-actin ratio (n=3 independent experiments, **P< 0.01 and *P<0.05 by unpaired Student's t test).

(D) Western blot for HIF-1α after 5 hours of hypoxia (1% O₂) in p75^NTR−/− MEFs infected with lentivirus expressing p75FL. β-Actin loading controls were performed on the same membrane. The ratio HIF1-α/β-actin is normalized to hypoxic WT cells.

(E) Immunocytochemical analysis of HIF-1α nuclear accumulation (WT normoxia, n=226; WT hypoxia, n=163; p75^NTR−/− normoxia, n=195; p75^NTR−/− hypoxia, n=240) after 5 hours of normoxia and hypoxia (1% O₂) in WT and p75^NTR−/− MEFs. Values are mean ± SD of the HIF1-α/β-actin ratio (n=2 independent experiments, *P<0.05 by unpaired Student's t test).

(F) Western blot for HIF-1α accumulation in nuclear and cytoplasmic fractions after 5 hours of normoxia or hypoxia (1% O₂) in WT and p75^NTR−/− MEFs. GAPDH and histone H3 (H3) were used as a loading control for cytoplasmic and nuclear fractions respectively. Representative western blot from n=2 independent experiments is shown. N, nuclear; NC, nucleo-cytoplasmic; C, cytoplasm.

(G) Real-time PCR analysis of GLUT1, PHD3 and VEGF expression in WT and p75^NTR−/− cells after 5 hours of normoxia and hypoxia (1% O₂). Values are mean ± SEM (GLUT1 and PHD3; MEFs, n=4; VEGF; CGNs, n=3 independent experiments, **P<0.01; *P<0.05 and ns, not significant, by one-way ANOVA). See also Figure S1.