Journal: Redox Biology

Article Title: PHB2 ameliorates ferroptosis and aortic aneurysm/dissection through NEDD4L-dependent ubiquitination of NCOA4

doi: 10.1016/j.redox.2026.104114

Figure Lengend Snippet: PHB2-evoked NCOA4 degradation through ubiquitination. (A and B). Representative Western blotting images and quantification of NCOA4 expression in VSMCs with or without PHB2 expression (n = 6 per group, p < 0.0001). C. Quantification of relative mRNA levels of NCOA4 in VSMCs with or without PHB2 overexpression (n = 6 per group). D. Representative Western blotting images depicting the effect of proteasome inhibitor (MG132) and lysosomal inhibitors (Pepstatin A + E64d) on NCOA4 protein levels in VSMCs with or without PHB2 overexpression. (E and F) Representative Western blotting images and quantification of NCOA4 protein stability in CHX-chase assays. G. Representative Co-IP images showing the ubiquitination of NCOA4 with or without PHB2 overexpression. H. Representative Co-IP images showing the ubiquitination of NCOA4 with or without PHB2 silencing. Mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, no significance.

Article Snippet: To determine whether protein degradation was mediated by the ubiquitin proteasome pathway, cells were treated with the proteasome inhibitor MG132 (10 μM; MedChemExpress) for 6 h before harvest.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Over Expression, Co-Immunoprecipitation Assay

Journal: Redox Biology

Article Title: PHB2 ameliorates ferroptosis and aortic aneurysm/dissection through NEDD4L-dependent ubiquitination of NCOA4

doi: 10.1016/j.redox.2026.104114

Figure Lengend Snippet: PHB2-induced regulation of NCOA4 ubiquitination through modulating NEDD4L activity. A. Bioinformatics prediction of interactions between NCOA4 and multiple E3 ubiquitin ligases using the UbiBrowser website ( http://ubibrowser.ncpsb.org.cn ). B. Endogenous NCOA4 and NEDD4L co-precipitated in VSMCs. C. Exogenous Flag-NCOA4 and HA-NEDD4L co-precipitated in VSMCs. D. Endogenous NCOA4 and PHB2 co-precipitated in VSMCs. E. Exogenous Flag-PHB2 and HA-NEDD4L co-precipitated in VSMCs. F. Representative Co-IP images showing that NEDD4L knockdown reduced PHB2-induced ubiquitination of NCOA4. G. Representative immunofluorescence photographs of co-localization of PHB2 and NCOA4 in VSMCs. H. Representative Western blotting images depicting subcellular localization of PHB2 and NEDD4L. I. Representative immunofluorescence photographs showing the effect of PHB2 overexpression on NEDD4L dimerization. J. Exogenous Co-IP assays of NEDD4L homodimerization via interaction between HA-NEDD4L and Flag-NEDD4L. K. Western blot analysis of NEDD4L dimer formation in vehicle versus PHB2-overexpressing groups.

Article Snippet: To determine whether protein degradation was mediated by the ubiquitin proteasome pathway, cells were treated with the proteasome inhibitor MG132 (10 μM; MedChemExpress) for 6 h before harvest.

Techniques: Ubiquitin Proteomics, Activity Assay, Co-Immunoprecipitation Assay, Knockdown, Immunofluorescence, Western Blot, Over Expression

Journal: iScience

Article Title: SplitTurboID mapping of dimeric protein phosphatase complex interactomes

doi: 10.1016/j.isci.2026.115195

Figure Lengend Snippet: c20orf27 is a novel PP1 interactor linked to mRNA splicing that also associates separately with an E3 ubiquitin ligase complex (A) Putative RVxF motif in wild-type c20orf27 and mutation of the hydrophobic residues that should abrogate PP1 binding. Homogenous distribution of GFP-c20orf27 throughout the cytoplasm in formaldehyde-fixed U2OS cells. (B) Biofluorescence complementation (BiFC) assays confirm the re-formation of functional YFP when its N- and C-terminal halves are tagged to wild-type c20orf27 and PP1. This association is lost for the KAGA mutant. For A-B, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision CORE system using a 60x/NA1.4 oil immersion objective. (C) Quantitative IP/MS comparing GFP-c20orf27 (H) to GFP (L) in HeLa cells identified significant enrichment (>2-fold) of PP1 and a CUL2 E3 ubiquitin ligase complex with c20orf27. (D) Validation of the co-immunoprecipitation of the ligase substrate-specifier APPBP2 with GFP-c20orf27. To assess the impact of proteasomal inhibition, the assay was carried out in both untreated cells and in cells treated with the proteasome inhibitor MG132 (10 μM for 1 h; confirmed by anti-ubiquitin staining of the lysates). Ponceau S staining of the membrane (right panel) confirmed equivalent total protein loading and immunoprecipitation. (E) Functionally grouped biological process networks generated using Cytoscape:ClueGO for high-confidence hits identified in a splitPP1:c20orf27 experiment overlapped with splitPP1:RRP1B and splitPP1:TPRN. (F) 2D heatmap graph compares the distribution of protein hits between the splitPP1:NIPP1 (green) and splitPP1:c20orf27 (magenta) datasets, based on their fractional enrichment in each. Arrows indicate the threshold of 0.75 above which proteins are considered to be enriched in a particular dataset. Lines indicate spliceosome components and hnRNP proteins that were enriched above threshold for splitPP1:NIPP1 and splitPP1:c20orf27, respectively (see Table in ). (G) Accumulation of proteins biotinylated by splitPP1:c20orf27 (magenta) at nuclear speckles (arrows) stained with anti-SC35-AF488 (green). U2OS cells co-expressing NTD-c20orf27 and CTD-PP1ɣ were incubated with 50 μM biotin for 4 h prior to formaldehyde fixation and streptavidin-AF647 staining. Enlarged panels show the individual anti-SC35-AF488 and biotin signals for the boxed region. (H) Localization of a pool of GFP-c20orf27 (green) at nuclear speckles (arrows) marked by anti-SC35-AF555 staining (magenta) in methanol-fixed U2OS cells. Enlarged panels show the individual GFP-c20orf27 and anti-SC35-AF555 signals for the boxed region. Additional accumulation of GFP-c20orf27 was observed at the perinuclear region (arrowhead). (I) Analysis of co-expressed mCh-c20orf27 and APPBP2-GFP in methanol-fixed U2OS cells shows that both are enriched at the perinuclear region (arrowheads), with additional accumulation in nuclear foci (arrows) observed for mCh-c20orf27. For G–I, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision Elite system using a 60x/NA1.4 oil immersion objective. See also .

Article Snippet: Monoclonal mouse anti-Ubiquitin , Santa Cruz , Cat#sc-8017; RRID: AB_628423.

Techniques: Ubiquitin Proteomics, Mutagenesis, Binding Assay, Functional Assay, Staining, Protein-Protein interactions, Biomarker Discovery, Immunoprecipitation, Inhibition, Membrane, Generated, Expressing, Incubation