Hypoxia-induced HIF-1α Regulation in MPNST

Cytoplasmic and nuclear extractions of MPNST cell lines were prepared separately using NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA). To inhibit the degradation of HIF-1α overexpressed under hypoxic conditions for 24 h, the cells were scraped and collected immediately after removal from the hypoxic chamber. Cellular extractions stimulated with chetomin or deferoxamine (DFO) for 24 h were also collected. Western blot analysis was performed as described previously [32 (link), 33 (link)] with the following primary antibodies: HIF-1α (1:1000, Abcam), β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), and lamin A/C (1:200, Santa Cruz). Immunoblotting of HIF-1α was performed with nuclear extraction as described previously [34 (link), 35 (link)].