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. 2024 Feb 22;15:1637. doi: 10.1038/s41467-024-45525-3

Fig. 6. ZNF598 undergoes regulatory K63-linked ubiquitination under mitochondrial stress.

Fig. 6

a Immunoblots showing ubiquitinated ZNF598 in HEK293T cells co-transfected with FLAG-ZNF598 and HA-Ub. Cells were treated with 2 μM Rotenone or 10 μM CCCP for 24 h followed by denaturing FLAG-IP. b Immunoprecipitation analysis of self-assembly of ZNF598 in cells co-transfected with GFP-ZNF598 and ZNF598-FLAG. c Effect of wild type ZNF598 and catalytically inactive mutant ZNF598 (ZNF598 CS) on GFP-P2A-Flag-K20-P2A-mKate2 stall reporter expression in HEK293T cells. d Effect of wild type ZNF598 and catalytically inactive ZNF598 (ZNF598 CS) on FLAG-GR80 expression in transfected HeLa cells. e Immunoblots showing ubiquitination of ZNF598 WT and inactive ZNF598 mutant (ZNF598 CS) in HEK293T cells co-transfected with FLAG-ZNF598 and HA-Ub. Cells were treated with 10 μM CCCP for 24 h followed by denaturing FLAG-IP. f Immunoblots showing effect of CCCP treatment (10 μM, 24 h) on ZNF598 ubiquitination in HEK293T cells co-transfected with Flag-ZNF598 and HA-Ub followed by denaturing IP and detection of Ub. The signals in the Flag-IP represent ubiquitinated ZNF598. g Immunoblots showing effect of CCCP treatment (10 μM, 24 h) on ZNF598 ubiquitination in HEK293T cells co-transfected with Flag-ZNF598 and HA-Ub-K63 or HA-Ub-K63R followed by denaturing IP and detection of Ub. All data are representative of at least three independent experiments. h Immunoblots showing effect of HA-Ub-K63 and HA-Ub-K63R OE on GFP-FLAG-K20-mCherry reporter expression in ZNF598 overexpressing HEK293T cells. i Cell viability measurement using CCK8 assay in HEK293T cells transfected with plasmids overexpressing ZNF598, Ub-K63, or Ub-K63R for 48 h followed by treatment with 20 μM CCCP (24 h). Data are representative of at least three biologically independent experiments (mean ± SD). Representative blot is from at least three independent experiments with similar results. p values were calculated by one-way ANOVA (Tukey’s test). Source data are provided as a Source Data file.