Fig. 4: Citrullination of HIF-1α disrupts the interaction of HIF-1α and VHL.

a HEK293T cells transfected with Flag-EV or Flag-HIF-1α plasmid together with PADI4 plasmid were cultured under normoxic or hypoxic conditions for 6 h, followed by immunoprecipitation analysis. The IP samples were derived from the same experiment, but different gels for PADI4, R698Cit, another for Flag were processed in parallel. b HEK293T cells transfected with HA-HIF-1α^WT or HA-HIF-1α^DM (double mutant, P402/564 A) plasmids together with PADI4 plasmids were cultured under normoxic or hypoxic conditions for 6 h, followed by immunoprecipitation analysis. The IP samples were derived from the same experiment, but different gels for PADI4, HA, and another for HIF-1α-OH were processed in parallel. c HEK293T cells were transfected with HA-EV or HA-HIF-1α plasmids together with psin-PADI4 plasmids with or without DFO in the presence of MG132 under normoxic condition for 6 h, followed by immunoprecipitation analysis. The IP samples were derived from the same experiment, but different gels for HA, another for HIF-1α-OH and another for R698Cit, PADI4 were processed in parallel. d Hep3B (upper panel) and HepG2 (lower panel) cells expressing NTC or PADI4 shRNAs were treated by hypoxia or DFO for 6 h, followed by western blotting analysis. e HEK293T cells transfected with EV or PADI4 together with HA-HIF-1α and Flag-VHL plasmids were cultured under normoxic or hypoxic conditions in the presence of 10 μM MG132 for 8 h, followed by immunoprecipitation analysis (upper panel). Quantification of Flag-VHL protein levels relative to HA-HIF-1α protein levels (lower panel). f Hep3B-Flag-EV or Flag-PADI4 cells infected with a virus expressing NTC or VHL shRNA were cultured under normoxic or hypoxic conditions for 6 h, followed by western blotting analysis. g RCC90 (VHL wild type) and RCC10 (VHL-null) cells expressing Flag-EV or Flag-PADI4 were cultured under normoxic or hypoxic conditions for 6 h, followed by western blotting analysis. h In vitro analysis of the interaction between purified GST-HIF-1α and His-VHL with increased amounts of His-PADI4. i HEK293T cells transfected with Flag-HIF-1α^WT or Flag-HIF-1α^R698A mutant and HA-VHL plasmids were cultured under normoxic or hypoxic conditions for 6 h, followed by immunoprecipitation analysis. j HEK293T cells transfected with Flag-HIF-1α^WT or Flag-HIF-1α^R698A mutant together with HA-Ub and PADI4 plasmids were cultured under normoxic or hypoxic conditions for 6 h, followed by immunoprecipitation analysis. The IP samples were derived from the same experiment, but different gels for PADI4, Flag, and another for HA were processed in parallel. k Diagram showing the mechanism by which PADI4 promotes the stability of HIF-1α. Immunoblots are representative of three independent experiments (a–j). Error bars denote the mean ± SEM (e). Statistical analyses were performed by ordinary one-way ANOVA with Turkey’s multiple comparisons test (e). Figure 4k created with BioRender.com is released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.