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. 2022 Apr 20;298(6):101961. doi: 10.1016/j.jbc.2022.101961

Figure 4.

Figure 4

The prolyl hydroxylase activity of EGLN1 is attenuated by SET7-mediated methylation on K297.A, Western blot analysis of endogenous EGLN1 and HIF1α expression in EGLN1-deficient or WT H1299 cells (EGLN1−/− or EGLN1+/+). B, Western blot analysis of exogenous Myc-HIF1α expression in EGLN1-deficient H1299 cells (EGLN1−/−) transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1α and WT FLAG-EGLN1 or the enzymatically deficient mutant (H313A), the methylation-mimic mutant (K297F). FLAG empty was used as a control. The relative intensities of Myc-HIF1α were determined by normalizing the intensities of Myc-HIF1α to the intensities of β-ACTIN. C, Western blot analysis of HIF1α hydroxylation with anti-hydroxy-HIF1α (Pro564) antibody in EGLN1-deficient H1299 cells (EGLN1−/−) transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1α and WT FLAG-EGLN1 or the enzymatically deficient mutant (H313A), the methylation-mimic mutant (K297F), followed by MG-132 (20 μM) treatment for 8 h. FLAG empty was used as a control. The relative intensities of HIF1α-OH/Myc-HIF1α were determined by normalizing the intensities of HIF1α-OH to the intensities of Myc-HIF1α. D, Western blot analysis of HIF1α hydroxylation with anti-Hydroxy-HIF1α (Pro564) antibody in HEK293T transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1α, FLAG-EGLN1, and HA-SET7 (HA empty was used as a control), followed by MG-132 (20 μM) treatment for 8 h. The relative intensities of HIF1α-OH/Myc-HIF1α were determined by normalizing the intensities of HIF1α-OH to the intensities of Myc-HIF1α. E, Western blot analysis of endogenous HIF1α expression in EGLN1-deficient H1299 cells (EGLN1−/−) transfected with indicated plasmid. The cells were transfected with WT FLAG-EGLN1 or the methylation-mimic mutant (K297F). FLAG empty was used as a control. The relative intensities of HIF1α were determined by normalizing the intensities of HIF1α to the intensities of GAPDH. F, Western blot analysis of HIF1α hydroxylation with anti-hydroxy-HIF1α (Pro564) antibody in EGLN1-deficient H1299 cells (EGLN1−/−) transfected with indicated plasmid. The cells were transfected with WT FLAG-EGLN1 or the methylation-mimic mutant (K297F) (HA empty was used as a control), followed by MG-132 (20 μM) treatment for 8 h. The relative intensities of HIF1α-OH/HIF1α were determined by normalizing the intensities of HIF1α-OH to the intensities of HIF1α. G, Western blot analysis of HIF1α hydroxylation with anti-hydroxy-HIF1α (Pro564) antibody in SET7-deficient or WT H1299 cells (SET7−/− or SET7+/+) treated with MG-132 (20 μM) for 8 h. The relative intensities of HIF1α-OH/HIF1α were determined by normalizing the intensities of HIF1α-OH to the intensities of HIF1α. H, Western blot analysis of exogenous Myc-HIF1α expression in HEK293T transfected with indicated plasmid. The cells were cotransfected with Myc-HIF1α, FLAG-EGLN1, and HA-SET7 (HA empty was used as a control), followed by CHX (50 μg/ml) treatment for indicated times. Myc empty was used as a control. The relative intensities of Myc-HIF1α were determined by normalizing the intensities of Myc-HIF1α to the intensities of β-actin. CHX, cycloheximide; EGLN, egg-laying defective nine; HA, hemagglutinin; HIF, hypoxia-inducible factor.