Western Blot Analysis of Protein Targets

Total protein extracts were isolated in lysis buffer as previously described [22 (link)]. Equal amounts of proteins (10–20 μg) were resolved using SDS-PAGE gels and transferred to PVDF Hybond-p membrane (GE Healthcare). Membranes were blocked with I-block (Life Technologies) for at least 2 hours, under rotation at RT. Membranes were then incubated overnight at 4°C under constant shaking with the following primary antibodies: HIF-1α (mouse, 1 : 250, BD Pharmingen), mTOR total, mTOR (S2448), P70S6K total, P70S6K (T389), AKT total, AKT (T308), AKT (S473), BAX, PARP, (all rabbit, 1 : 1000, Cell Signaling Technologies), and β-actin (mouse, 1 : 10000, Sigma Aldrich) as loading control. Membranes were next incubated with peroxidase-labeled goat anti-rabbit IgG or goat anti-murine IgG (both 1 : 50.000 in I-block from Sigma Aldrich) for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and exposed to Hyperfilm MP (GE Healthcare).