Figure 2

TRIM25 interacts with HIF-1α to stabilize HIF-1α protein and cause HIF-1α accumulation in the nucleus.A, Co-IP of ectopically expressed TRIM25 interacting with ectopically expressed HIF-1α in HEK293T cells. B, endogenous IP of TRIM25 interacting with endogenous HIF-1α in U251 cells under hypoxia (1% O₂) for 4 h. C, the interaction of bacterially expressed GST-HIF-1α and bacterially expressed His-TRIM25. D, IB of Myc-HIF-1α in HEK293T cells transfected with Myc-HIF-1α together with Flag-empty vector or increasing amounts of Flag-TRIM25 expression plasmids. E, IB of endogenous HIF-1α in H1299 cells transfected with Flag-empty vector or increasing amounts of Flag-TRIM25 expression plasmids for 18 h, followed by treatment with normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. F, IB of endogenous HIF-1α in U251 cells transfected with Flag-empty vector or increasing amounts of Flag-TRIM25 expression plasmids for 18 h, followed by treatment with hypoxia (1% O₂) for 4 h. G, IB of endogenous HIF-1α in wild-type and TRIM25-deficient U251 cell lines (#1 and #2) treated with or without CoCl₂ (200 μM) for 6 h. H, IB of endogenous HIF-1α in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cell lines (#1, #2 and #3) under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. I, IB of endogenous HIF-1α in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cell lines cultured under hypoxia (1% O₂) for 4 h. CHX was then added to inhibit new protein synthesis with a final concentration of 50 μg/ml at the indicated time points. J, IB of endogenous HIF-1α in cytosolic and nuclear fractions in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cells cultured under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. K, Confocal microscopy image of endogenous HIF-1α in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cells under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. Scale bar = 50 μm.