Fig. 5: OTUB1 stabilizes HIF-1α by inhibiting ubiquitination of HIF-1α. | Cell Death & Disease

Fig. 5: OTUB1 stabilizes HIF-1α by inhibiting ubiquitination of HIF-1α.

From: OTUB1 augments hypoxia signaling via its non-canonical ubiquitination inhibition of HIF-1α during hypoxia adaptation

Fig. 5

A Immunoblotting of exogenous Myc-HIF-1α expression in HEK293T cells transfected with an increasing amount of Flag-OTUB1 expression plasmid. B Immunoblotting of exogenous Myc-HIF-1α expression in H1299 cells transfected with an increasing amount of Flag-OTUB1 expression plasmid. C Immunoblotting of endogenous HIF-1α expression in WT or two OTUB1-deficient H1299 cell lines (#1 and #2) cultured under normoxia (21% O2) or hypoxia (1% O2) for 4 h. D Immunoblotting of exogenous HA-HIF-1α-DM (encoding the double mutant of HIF-1α [P402A/P564A]) expression in HEK293T cells transfected with an increasing amount of Flag-OTUB1 expression plasmid. E Immunoblotting of exogenous HA-HIF-1α-TM (encoding the triple mutant of HIF-1α [P402A/P564A/N803A]) expression in HEK293T cells transfected with an increasing amount of Flag-OTUB1 expression plasmid. F Immunoblotting of endogenous HIF-1α expression in WT or two OTUB1-deficient H1299 cell lines treated with DMSO (as a control) or DMOG (1 mM) for 6 h. G Immunoblotting of endogenous HIF-1α expression in WT or OTUB1-deficient H1299 (OTUB1+/+ or OTUB1−/−) cells treated with an increasing amount of FG4592 for 6 h. H Immunoblotting of endogenous HIF-1α expression in WT or OTUB1-deficient H1299 (OTUB1+/+ or OTUB1−/−) cells treated with an increasing time of FG4592 (100 μM). I The relative intensities of HIF-1α in G determined by normalizing the intensities of HIF-1α to the intensities of β-actin. J The relative intensities of HIF-1α in H determined by normalizing the intensities of HIF-1α to the intensities of β-actin. K HIF-1α ubiquitination in HEK293T cells transfected with indicated plasmids for 24 h. L Endogenous HIF-1α ubiquitination in H1299 cells transfected with indicated plasmids and cultured under normoxia (21% O2) for 24 h, followed by MG-132 (20 μM) treatment for 6~8 h. M Endogenous HIF-1α ubiquitination in WT or OTUB1-deficient H1299 (OTUB1+/+ or OTUB1−/−) cells cultured under normoxia (21% O2) and treated with MG-132 (20 μM) for 6~8 h.

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