Protein Expression Analysis by Western Blot

The levels of HIF-1α (Abcam; Cambridge, UK), p-P70S6K(Thr389) (New England Biolabs; Hitchin, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Heidelberg, Germany) were determined by Western blot analysis as previously described [45 (link), 46 (link)]. Following treatment and incubation for 24 h cells were collected as described above. Cell extracts were loaded on acrylamide mini gels and separated proteins were transferred onto PVDF Immobilon-P^® membranes then blocked in PBS-Tween containing 5% (w/v) non-fat dry milk. Primary antibodies were incubated overnight at 4°C. Secondary peroxidase-conjugated antibodies (GE healthcare, Buckinghamshire, UK) were added in PBS-Tween/milk. Membranes were exposed to chemiluminescent HRP substrate (GE Healthcare Buckinghamshire, UK) and visualised using the ChemiDoc-IT Imaging System (UVP, Bio-Rad, Hertfordshire, UK).