ha tagged ubiquitin plasmids  (Addgene inc)

Journal: Redox Biology

Article Title: Inhibition of SDE2 promotes autophagy-dependent ferroptosis in multiple myeloma

doi: 10.1016/j.redox.2026.104007

Figure Lengend Snippet: Molecular interaction between SDE2 and ATG5. (A) Molecular docking prediction illustrating the interaction between ATG5 and SDE2. (B) Box plot showing significantly elevated ATG5 expression in plasma cells from MM patients compared to healthy controls. Data were obtained from the TCGA database. (C) Kaplan–Meier survival analysis of multiple myeloma (MM) patients stratified by combined expression levels of ATG5 and SDE2. (D) Western blot analysis demonstrating the effect of SDE2 knockdown on ATG5 protein levels in OPM-2 and KMS-11 cells. (E–F) Co-immunoprecipitation (Co-IP) assays in KMS-11 cells using antibodies targeting SDE2 to pull down ATG5 (E) and antibodies targeting ATG5 to pull down SDE2 (F), confirming a direct interaction between the two proteins. (G) HEK 293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2. Immunoprecipitation using anti-Flag antibodies was followed by immunoblotting with anti-Myc (ATG5) and anti-Flag (SDE2) antibodies, validating the interaction between exogenous SDE2 and ATG5. (H) Western blot analysis of ATG5 degradation in SDE2-overexpressing cells treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) in the presence of chloroquine (CQ) or MG132. (I) Western blot analysis showing that treatment with MG132 rescues ATG5 degradation in SDE2-overexpressing cells. (J) Schematic representation of full-length and truncation constructs of SDE2. (K) Co-immunoprecipitation of HA-SDE2 variants with Flag-tagged ATG5 in HEK293T cells. Only full-length and 1–300 aa fragment of SDE2 retained the ability to bind ATG5. (L) Cell lysates from SDE2-overexpressing cells (wild-type, Δ1, and Δ2 mutants) were immunoprecipitated with anti-ATG5 antibodies and immunoblotted with anti-Ub and anti-ATG5 antibodies to assess ATG5 ubiquitination levels. (M) HEK 293T cells were co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged ATG5, and Flag-tagged SDE2 (wild-type and Δ1 mutant). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, demonstrating that the SDE2 UBL domain mediates ATG5 ubiquitination. (N) Co-IP analysis of the interaction between ATG5 and the SDE2-Δ1 mutant. HEK293T cells were co-transfected with Myc-tagged ATG5 and Flag-tagged SDE2-Δ1 plasmids as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Myc and anti-Flag antibodies. Input blots confirmed protein expression levels. (O) M. SDE2-Δ1 fails to promote ATG5 degradation in KMS-11 cells. Cells were transfected with SDE2-Δ1 and treated with or without the proteasome inhibitor MG132 (10 μM, 6 h). (P) HEK 293T cells were co-transfected with Myc-tagged ATG5, Flag-tagged SDE2, and HA-tagged ubiquitin constructs (wild-type, Lys48-only, or Lys63-only). Immunoprecipitation using anti-Myc antibodies was followed by immunoblotting with anti-HA and anti-Myc antibodies, confirming that SDE2 facilitates Lys48-linked ubiquitination of ATG5. (Q) Western blot analysis showing ATG5 and SDE2 levels in control and SDE2-overexpressing KMS-11 cells co-expressing wild-type ubiquitin (Ub WT) or ubiquitin with a Lys48-to-Arg mutation (Ub Lys48R) after 72 h of culture. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Ubiquitination Assay Plasmids: HA-tagged ubiquitin plasmids (wild-type: #17608; K48-linked: #17605; K63-linked: #17606) were obtained from Addgene.

Techniques: Expressing, Clinical Proteomics, Western Blot, Knockdown, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Ubiquitin Proteomics, Mutagenesis, Control

Journal: bioRxiv

Article Title: IFI207 promotes antiviral responses by modulating STING ubiquitination and degradation

doi: 10.64898/2026.03.05.709838

Figure Lengend Snippet: IFI207 reduces K63-linked ubiquitination on STING following DMXAA stimulation. A) BMDMs from the indicated mice were treated with 100 µg/mL DMXAA for 1 hr. An anti-K63-linkage-specific polyubiquitin antibody was used to immunoprecipitate cell extracts. The immunoprecipitates were then analyzed by western blotting using anti-STING and anti-tubulin antibodies. Shown to the right is quantification of 3 independent experiments ± SD. Two-way ANOVA was used to determine significance. **, P ≤0.03; ns, not significant. B) HEK293T cells were transfected with either HA-tagged Ub-WT or -K63 expression plasmids along with Flag-tagged STING and V5-tagged IFI207. 24 hr after transfection, the cells were treated with 100 μg/ml DMXAA for 2 hr. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. To quantify, the ubiquitin signal was normalized to the STING signal and then the EV (4 hr) sample was set to 1. Shown below is quantification of the average of 3 independent experiments ± SD. Two-way ANOVA was used to determine significance. *, P ≤0.01; ns, not significant. C) HEK293T cells were transfected with HA-tagged Ub-63 plasmids along with Flag-tagged STING and V5-tagged IFI207. 24 hr after transfection, the cells were pre-treated with TAK243 for 30 mins and then stimulated with DMXAA for 4 hr. Immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. Shown to the right is quantification of the average of 3 independent experiments ± SD. Two-way ANOVA was used to determine significance. **, P ≤0.0008; ns, not significant.

Article Snippet: The HA-tagged Ub-WT HA-Ub (#17608) and HA-Ub (K63 only) (#17606) plasmids were from Addgene.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Expressing, Immunoprecipitation