Journal: Signal Transduction and Targeted Therapy
Article Title: Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis
doi: 10.1038/s41392-024-01763-x
Figure Lengend Snippet: Phosphorylation of Trim25 at S100 is required for ITPKB ubiquitination. a Phosphorylation proteomics analysis of Trim25 in primary and recurrence GBM. b The phosphorylation level of Trim25 S100 site in GBM tissue. c Dephosphorylation of the E3 ligase Trim25 resulted in a more pronounced alleviation of ITPKB ubiquitination in a cell-free system compared to mock-treated Trim25. Dephosphorylated or mock-treated Trim25 was incubated with immunopurified Flag-ITPKB, ubiquitin, recombinant E1 (Uba1), and E2 (UbcH5b). ITPKB ubiquitination was then determined using a ubiquitination assay. d The interaction between ITPKB and Trim25 was affected by the Trim25 S100D phosphomimetic mutant and S100A dephosphorylation mutant. e The ubiquitination of ITPKB was altered when the S100 site was mutated to Asp (S100D) or Ala (S100A). HEK293T cells were co-transfected with the respective plasmids and treated with MG132 for 3 h after 48 h of transfection. Cell lysates were immunoprecipitated and blotted with specific antibodies for analysis. Statistical significance is shown as: * p < 0.01
Article Snippet: The E3 ligase complex was then mixed with 2 μg Flag-ITPKB purified from HEK293T cells and reacted with 15 μg wild-type ubiquitin (Boston Biochem, U-100H-10M), 550 ng of E1 (Uba1, Boston Biochem, E-305-025), and 850 ng of E2 (UbcH5b, Boston Biochem, E2-622-100).
Techniques: Phospho-proteomics, Ubiquitin Proteomics, De-Phosphorylation Assay, Incubation, Recombinant, Mutagenesis, Transfection, Immunoprecipitation
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