Fig. 2: CHK2 stabilizes HIF-1α by diminishing ubiquitination of HIF-1α.
From: ROS-ATM-CHK2 axis stabilizes HIF-1α and promotes tumor angiogenesis in hypoxic microenvironment
A Western blotting of exogenous HA-HIF-1α and Flag-tagged CHK2. HA-tagged HIF-1α (3 μg) and Flag-tagged CHK2 (1 μg, 3 μg) was transiently transfected into H1299 cells and then exposed to 1% O2. B Western blotting of exogenous HA-HIF-1α and CHK2. HA-tagged HIF-1α was transiently transfected into HEK293 and HEK293-CHK2-KO cells under hypoxic microenvironment. MG132 and CQ was then added to above cells respectively or together as indicated. C Endogenous HIF-1α ubiquitination in HEK293 and HEK293-CHK2-KO cells under hypoxia (1% O2) for 6 h. Exogenous HA-HIF-1α ubiquitination under normoxia (D) and hypoxia (E) in HEK293-CHK2-KO cells transiently transfected with indicated plasmids. F Western blotting of exogenous double mutant HIF-1α protein at both hydroxylation sites P402A/P564A (HA-HIF-1α-DM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia. G Western blotting of exogenous triple mutant HIF-1α protein at P402A/P564A/N803A (HA-HIF-1α-TM). HEK293 cells were transfected with an increasing amount of Flag-CHK2 expression plasmid and exposed to hypoxia.
