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. Author manuscript; available in PMC: 2012 Aug 16.

Published in final edited form as: Exp Cell Res. 2010 Mar 19;316(10):1706–1715. doi: 10.1016/j.yexcr.2010.03.005

A. nL-8 cells (1 × 10⁶ cells each) were treated either with the PI3K inhibitor LY294002 (25 μM) or the mTOR inhibitor rapamycin (10 nM) or vehicle and incubated under hypoxic conditions for 6 h, following which total protein was isolated and tested for the levels of HIF-1α by immunoblotting (30 μg total protein per sample). Shown here is a representative result from three independent experiments performed under essentially identical conditions. B. nL-8 and neo cells were transiently transfected either with a PTEN expression construct or with the empty vector. PTEN transfected cells (1 × 10⁶ cells per incubation) were treated with LY294002 (25 μM) or vehicle (DMSO) and incubated under hypoxia for 6 h. Total protein (30 μg total protein per sample) harvested after incubation was tested for the levels of PTEN, HIF-1α, GLUT-1, p-Akt, and Akt by immunoblotting. Results shown here are from a representative experiment of n = 3; sample from one experiment was used for each protein tested and the membrane was reprobed for loading controls, i.e. actin and total Akt.

A. nL-8 cells (1 × 10⁶ cells each) were treated either with the PI3K inhibitor LY294002 (25 μM) or the mTOR inhibitor rapamycin (10 nM) or vehicle and incubated under hypoxic conditions for 6 h, following which total protein was isolated and tested for the levels of HIF-1α by immunoblotting (30 μg total protein per sample). Shown here is a representative result from three independent experiments performed under essentially identical conditions. B. nL-8 and neo cells were transiently transfected either with a PTEN expression construct or with the empty vector. PTEN transfected cells (1 × 10⁶ cells per incubation) were treated with LY294002 (25 μM) or vehicle (DMSO) and incubated under hypoxia for 6 h. Total protein (30 μg total protein per sample) harvested after incubation was tested for the levels of PTEN, HIF-1α, GLUT-1, p-Akt, and Akt by immunoblotting. Results shown here are from a representative experiment of n = 3; sample from one experiment was used for each protein tested and the membrane was reprobed for loading controls, i.e. actin and total Akt.