Hypoxic Activation of K-Ras Figure 2. Activation of Ras by hypoxia is dependent on c-Src activation. A, Caco2 and HT29 cells were incubated in hypoxic conditions for the indicated times, and Western blotting for phospho-Src416 and total Src was performed. b-actin confirmed equal loading. Densitometry values are expressed as fold change compared with control values normalized to 1. B, Caco2 cells were pretreated with the Src inhibitor PP2 (20 mM) or DMSO for 1 hour (left panel), or transiently transfected with c-Src specific siRNA constructs or a non targeting control (right panel), before incubation in hypoxic or normoxic conditions for 4 hours. Activated Ras was pulled down and SDS-PAGE was performed using a Ras-GTP specific antibody. Total Ras confirmed equal loading. Densitometry values are expressed as fold change compared with control values normalized to 1. C, Lysates of Caco2pEVX and Caco2SrcY527F cells were immunostained for p-Src416 and total Src and also used for a Ras activation assay. Densitometry values for Ras-GTP are expressed as fold change compared with control values normalized to 1. D, left panel, Control cells and cells pretreated with NAC (20 mM) for 20 minutes were exposed for 10 minutes to H2O2 (5 mM). Lysates were then immunoblotted for p-Src416 and total Src. b-actin confirmed equal loading. Middle and right panels, Control Caco2 cells and cells pretreated with NAC (20 mM) for 1 hour were incubated in hypoxia for 4 hours. Western blotting for phospho-Src416 (middle panel) and a Ras activating assay (right panel) were then performed. Densitometry values are expressed as fold change compared with control values normalized to 1. E, Lysates of control Caco2 cells and cells incubated in hypoxia for the indicated times were subjected to Western blotting to detect p-EGFR (Tyr1068) and total EGFR protein levels. Treatment with EGF (100 ng/mL) served as a positive control. doi:10.1371/journal.pone.0010966.g002 PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e10966