Journal: Journal of Orthopaedic Translation
Article Title: TRIM59 alleviates neuronal ferroptosis and promotes functional recovery after spinal cord injury by mediating ubiquitination and degradation of ANXA2
doi: 10.1016/j.jot.2026.101070
Figure Lengend Snippet: TRIM59 enhances the ubiquitination and degradation of ANXA2. (A) Examination of ANXA2 and TRIM59 in Lv-shTRIM59-transfected neuronal cells with or without MG132 proteasome inhibitor treatment (n = 3); (B) Flag-tagged TRIM59 (WT or C30A mutant) was introduced into HEK 293 T cells, and cell lysates were examined by immunoblotting with anti-ANXA2 and anti-Flag (n = 3); (C, D) ANXA2 protein stability was evaluated following TRIM59 knockdown in HEK 293 T cells (n = 3); (E, F) ANXA2 protein stability was determined following TRIM59 overexpression in HEK 293 T cells (n = 3); (G, H) Lysates from neuronal cells transfected with Lv-shCtrl or Lv-shTRIM59, treated with MG132 prior to harvesting, were subjected to immunoprecipitation and detected with the specified antibodies (n = 3); (I, J) HEK 293 T cells transfected with HA-tagged Ub, Myc-tagged ANXA2, and Flag-tagged TRIM59 (WT or C30A) were immunoprecipitated and examined by anti-Myc, anti-HA, and anti-Flag western blotting (n = 3); (K, L) Spinal cord lysates from AAV-Con or AAV-TRIM59-injected mice were immunoprecipitated with anti-ANXA2, and Ub-ANXA2 was detected by western blot with anti-Ub and anti-ANXA2 antibodies (n = 6); (M) Quantification of ANXA2 ubiquitination in HEK 293 T cells co-transfected with Flag-TRIM59, Myc-ANXA2, and HA-Ub or K48-only or K63-only plasmids (n = 3); (N) HEK 293 T cells transfected with ubiquitin WT or K48R, cultured with shCtrl or shTRIM59 for 72 h, and examined by immunoblotting with anti-ANXA2 and anti-TRIM59 (n = 3). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
Article Snippet: The HEK 293 T cell line, acquired from Procell Life Science and Technology Co. (CL-0005, Wuhan, China), was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Waltham, MA) fortified with fetal bovine serum (10% FBS; SERANA, Brandenburg, Germany).
Techniques: Ubiquitin Proteomics, Transfection, Mutagenesis, Western Blot, Knockdown, Over Expression, Immunoprecipitation, Injection, Cell Culture
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