Journal: Redox Biology
Article Title: A LNK–CBL–HNRPA2B1–GPX4 signaling axis mediates dopaminergic neuron vulnerability to ferroptosis in Parkinson's disease
doi: 10.1016/j.redox.2026.104039
Figure Lengend Snippet: LNK-dependent phosphorylation and nuclear translocation of CBL is required for HNRPA2B1 ubiquitination (A) Endogenous co-IP assay in SH-SY5Y cells showing the interaction between LNK and CBL. (B) Representative immunofluorescence of HNRPA2B1 (green) and CBL (WT or Y731F, red) in MPP + -treated SH-SY5Y cells (1 mM, 24 h). Nuclei are stained with DAPI (blue). Scale bars, 20 μm. (C) Co-IP assay showing that LNK knockdown reduces the interaction between endogenous CBL and HNRPA2B1 in SH-SY5Y cells following MPP + treatment. (D) Co-IP assay showing that LNK overexpression enhances the interaction between endogenous CBL and HNRPA2B1 in SH-SY5Y cells following MPP + treatment. (E) Co-IP assay demonstrating that the CBL-HNRPA2B1 interaction is phosphorylation-dependent. Lysates from LNK-overexpressing SH-SY5Y cells following MPP + treatment were treated with or without λ-phosphatase prior to IP. (F) Subcellular fractionation and immunoblot analysis of protein levels in cytoplasmic and nuclear fractions from shNC and shLNK cells treated with or without MPP + . Right: Corresponding quantification of band intensities (n = 3). (G) Co-IP assay in HEK293T cells. His-tagged proteins (WT CBL or Y731F mutant) were immunoprecipitated (IP: His) from lysates of cells co-transfected with the indicated constructs. IB for Flag-HNRPA2B1. (H) Reciprocal co-IP assay in HEK293T cells. Flag-HNRPA2B1 was immunoprecipitated (IP: Flag) from lysates of cells co-transfected with the indicated constructs. IB for His-CBL. (I) Ubiquitination assay. HEK293T cells were co-transfected with Flag-HNRPA2B1, HA-Ub-K27, and the indicated LNK and CBL constructs. HNRPA2B1 polyubiquitination was assessed by IP with an anti-Flag antibody followed by IB with an anti-HA antibody. Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with Tukey's post-hoc test (F). ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant.
Article Snippet: Microdissected substantia nigra and striatum tissues or cells were homogenized in ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with a protease and phosphatase inhibitor cocktail (MedChemExpress, Monmouth Junction, NJ, USA).
Techniques: Phospho-proteomics, Translocation Assay, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Knockdown, Over Expression, Fractionation, Western Blot, Mutagenesis, Immunoprecipitation, Transfection, Construct
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