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

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A–C) In vitro auto-ubiquitination assays using (A) MBP-PUB32 FL compared to catalytic mutants MBP-PUB32 FL-W768A and MBP-PUB32 ΔUbox ; (B) His 6 -MBP-PUB33 FL compared to His 6 -MBP-PUB33 FL-W796A and His 6 -MBP-PUB33 ΔUbox ; (C) MBP-PUB50 FL compared to MBP-PUB50 ΔUbox . Reaction mixtures included ubiquitin, ubiquitin activating protein 1 (UBA1) and ubiquitin conjugating protein 8 (UBC8). Western blots were probed with antibodies against ubiquitin (anti-Ub) or maltose binding protein (anti-MBP); protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). (D-E) In vitro auto-phosphorylation assays of His 6 -MBP-PUB33 FL , His 6 -MBP-PUB33 KD , MBP-PUB32 FL , MBP-PUB32 KD , MBP-PUB50 FL , MBP-PUB50 KD , and His 6 -MBP-PUB51 FL ( D ) and His 6 -MBP-PUB33 FL , MBP-PUB33 FL-D603A , His 6 -MBP-PUB33 KD , and MBP-PUB33 KD-D603A ( E ). Autoradiographs (Autorad) show incorporation of γ-³²P, and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). Asterisks indicate the respective proteins. All assays were repeated at least three times with similar results by TD.

Article Snippet: For auto-ubiquitination assays, 2 μg of recombinant E3 ligase and 0.075 μg of HA-tagged ubiquitin (R&D Systems; U-110) were incubated in 40 μL of reaction buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 25 mM KCl, 0.33 mM DTT, 1.5 mM ATP, 200 ng E1 (UBA1; AT2G30110), and 450 ng E2 (UBC8; AT5G41700) for the indicated times.

Techniques: In Vitro, Ubiquitin Proteomics, Western Blot, Binding Assay, Staining, Phospho-proteomics

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) Schematic representation of PUB33 protein domains and truncated variants: PUB33 FL indicates the full-length protein; PUB33 CT indicates the kinase and U-box domains and removal of the entire N-terminus; PUB33 ΔUbox indicates removal of the U-box domain; PUB33 KD indicates only the kinase domain; PUB33 αHelix indicates only the α-helical domain. Exact amino acid boundaries for all variants can be found in Supplemental Table S2 . Schematic created by TD. (B-D) In vitro autophosphorylation assays of PUB33 variants as indicated. Autoradiography (Autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). Asterisks in B indicate expected proteins of interest. Note that the first and fourth lanes of the CBB-stained gel in D display characteristic ubiquitination smears corresponding to PUB33 FL or PUB33 CT, , indicating that the ubiquitin ligase is active. Assays conducted at least three times by TD with similar results. (E) In vitro trans-phosphorylation assay between His 6 -MBP-PUB33 FL and His 6 -MBP-PUB33 KD and the universal substrate histone H3S. Autorad indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB. Note that the signal from H3S is so strong that it masks the signal from His 6 -MBP-PUB33 KD when imaged together. Boxes indicate that some lanes were removed digitally for clarity but everything shown is from a single gel. Assays conducted at least three times by TD with similar results. (F) Possible configuration of a PUB33 homomer, as predicted by AlphaFold2. The colours match the labels in A. Average predicted Local Distance Difference Test (pLDDT) score for the PUB33 monomer is is 66.52 and the predicted template modeling (pTM) and interface pTM (ipTM) scores for the PUB33 homomer are 0.47 and 0.45, indicating low-to-moderate confidence in both the overall model and the predicted interface. Analysis by TD. (G) Yeast 2-hybrid between the indicated PUB33 variants. EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. DB indicates constructs translationally fused to the Locus for X-Ray Sensitivity A (LexA) DNA binding domain; AD indicates constructs translationally fused to the B42 transcriptional activation domain. Assays conducted by IK at least three times with similar results.

Article Snippet: For auto-ubiquitination assays, 2 μg of recombinant E3 ligase and 0.075 μg of HA-tagged ubiquitin (R&D Systems; U-110) were incubated in 40 μL of reaction buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 25 mM KCl, 0.33 mM DTT, 1.5 mM ATP, 200 ng E1 (UBA1; AT2G30110), and 450 ng E2 (UBC8; AT5G41700) for the indicated times.

Techniques: In Vitro, Autoradiography, Staining, Ubiquitin Proteomics, Phospho-proteomics, Plasmid Preparation, Control, Transformation Assay, Construct, Binding Assay, Activation Assay

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) In vitro auto-ubiquitination assays comparing His 6 -MBP-PUB33 FL with MBP-PUB33 FL-D603A . The reaction mixture included ubiquitin, ubiquitin activating protein 1 (UBA1) or ubiquitin conjugating protein 8 (UBC8). Western blots were probed with antibodies against ubiquitin (anti-Ub) and maltose-binding protein (anti-MBP) and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB). These assays were repeated more than three times with similar results by TD. (B) PUB33 phosphopeptides detected following in vitro His 6 -MBP-PUB33 FL autophosphorylation. Each peptide was identified in at least 2/3 independent replicates and was absent in control samples without ATP. Phosphosites are indicated in bold, with positions shown on the right. The schematic below indicates their location on PUB33 FL ; colours indicate the domains of PUB33 as defined in . Kinase assays were performed by TD; trypsin digestion and LC-MS/MS analysis were performed by MCRG. (C) Multiple sequence alignment of the regions including the PUB33 phosphosites across the PUB-VI/RLCK-IXb proteins. Analysis by TD. (D-E) In vitro auto-ubiquitination assays comparing His 6 -MBP-PUB33 FL with the indicated variants. Reaction mixtures include UBA1 and UBC8, with (+) and without (-) ubiquitin for the indicated times. Western blots were probed with anti-Ub and anti-MBP, and protein loading is indicated by post-staining with CBB. These assays were repeated more than three times with similar results by TD. (F) In vitro auto-phosphorylation assays comparing His 6 -MBP-PUB33 FL to PUB33 FL-T333A and PUB33 FL-T333D . Autoradiography (Autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB. These assays were repeated more than three times with similar results by TD. (G) Alignment of the predicted coiled-coil domain of the PUB33 homomer in its unphosphorylated (magenta) and phosphorylated at T333 (grey) configurations, as predicted by AlphaFold3. Although we noticed a modest shift in this region, the predicted PUB33 and PUB33-pT333 homomers are excellently aligned overall, with a sequence alignment score of 2264.2 and a root mean square deviation of 0.713 Å. Analysis by JM. (H) Yeast 2-hybrid between the indicated PUB33 variants. EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. Assays conducted by IK at least three times with similar results.

Article Snippet: For auto-ubiquitination assays, 2 μg of recombinant E3 ligase and 0.075 μg of HA-tagged ubiquitin (R&D Systems; U-110) were incubated in 40 μL of reaction buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 25 mM KCl, 0.33 mM DTT, 1.5 mM ATP, 200 ng E1 (UBA1; AT2G30110), and 450 ng E2 (UBC8; AT5G41700) for the indicated times.

Techniques: In Vitro, Ubiquitin Proteomics, Western Blot, Binding Assay, Staining, Control, Liquid Chromatography with Mass Spectroscopy, Sequencing, Phospho-proteomics, Autoradiography, Plasmid Preparation, Transformation Assay

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) Unique PUB32 peptides identified by mass spectrometry following immunoprecipitation of PUB33-GFP from Col-0/35S:PUB33-GFP line #9-4. Peptides were present in 3/3 PUB33-GFP samples and 0/3 Col-0 samples; see Supplementary Table S2 for more details. Immunoprecipitation performed by TD; sample processing and analysis performed by MCRG. (B,F) Yeast 2-hybrid between PUB32 FL and PUB33 FL (B) and PUB33 FL variants (F) . EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. DB indicates constructs translationally fused to the Locus for X-Ray Sensitivity A (LexA) DNA binding domain; AD indicates constructs translationally fused to the B42 transcriptional activation domain. Assays conducted by IK at least three times with similar results. (C) Possible configuration of a PUB32:PUB33 homomer, as predicted by AlphaFold2. The colours match the labels in A. Average predicted Local Distance Difference Test (pLDDT) score for the PUB32 monomer is 75.31 and the pLDDT score for the PUB33 monomer is 77.9. The predicted template modeling (pTM) and interface pTM (ipTM) scores for the PUB32:PUB33 heteromer are 0.50 and 0.49, indicating low-to-moderate confidence in both the overall model and the predicted interface. Analysis by TD. (D) In vitro auto-ubiquitination assays comparing 2 μg His 6 -MBP-PUB33 FL in the presence of increasing amounts of MBP-PUB32 FL . Reaction mixtures include UBA1, UBC8, and ubiquitin. Western blots were probed with anti-Ub and anti-MBP, and protein loading is indicated by post-staining with Coomassie Brilliant Blue (CBB) R250. These assays were repeated more than three times with similar results by TD. (E) In vitro auto-phosphorylation assays comparing 2 μg His 6 -MBP-PUB33 FL in the presence of increasing amounts of MBP-PUB32 FL . Autoradiography (Autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB G250. Assays conducted at least three times by TD with similar results.

Article Snippet: For auto-ubiquitination assays, 2 μg of recombinant E3 ligase and 0.075 μg of HA-tagged ubiquitin (R&D Systems; U-110) were incubated in 40 μL of reaction buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 25 mM KCl, 0.33 mM DTT, 1.5 mM ATP, 200 ng E1 (UBA1; AT2G30110), and 450 ng E2 (UBC8; AT5G41700) for the indicated times.

Techniques: Mass Spectrometry, Immunoprecipitation, Plasmid Preparation, Control, Transformation Assay, Construct, Binding Assay, Activation Assay, In Vitro, Ubiquitin Proteomics, Western Blot, Staining, Phospho-proteomics, Autoradiography

Journal: bioRxiv

Article Title: Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

doi: 10.64898/2026.02.10.704836

Figure Lengend Snippet: (A) Yeast 2-hybrid between the N-terminal domain of RipV1 (RipV1 NT ) and PUB32 FL , PUB33 FL , and the indicated PUB33 variants. EV is the empty vector control. Growth was assessed on SD media lacking His, Leu, Trp, and Uracil (-HLWU) with the addition of X-galactosidase (X-gal) as markers for protein:protein interaction or on SD-HWU as a transformation control. DB indicates constructs translationally fused to the Locus for X-Ray Sensitivity A (LexA) DNA binding domain; AD indicates constructs translationally fused to the B42 transcriptional activation domain. Assays conducted by JC and IK at least three times with similar results. (B-D) In vitro trans-ubiquitination of PUB33 FL-W796A (B) and PUB32 FL by RipV1 FL (C) ; or RipV1 C452A by PUB33 FL (D) . Reaction mixtures include UBA1, UBC8, and ubiquitin. Western blots were probed with anti-Ub and anti-MBP, and protein loading is indicated by post-staining Coomassie Brilliant Blue (CBB) R250. These assays were repeated more than three times with similar results by TD. (E) In vitro trans-phosphorylation assay between PUB33 FL or PUB33 KD and RipV1, compared to the kinase-dead variants containing D603A. Autoradiography (autorad) indicates γ-³²P incorporation, and protein loading is indicated by post-staining with CBB G250. Asterisks indicate proteins of interest. Assays were repeated 3 times by TD. (F) Cell death induced by transient expression of RipV1 FL -FLAG in the presence or absence of PUB33-HA or free GFP in N. benthamiana . False color photographs were taken at 3 days post infiltration (dpi); the circles indicate the infiltrated area. Dark areas indicate cell death. Quantum yield of chlorophyll (QY) measurements were also taken at 3 dpi and are summarized in the lower histogram. Values are quantum yield (Fv/Fm) from 3 independent experiments (n=9 leaf discs per experiment). The midline represents the median; interquartile ranges are represented by the boxes, and maximum and minimum values are represented by the whiskers. Significantly different groups are labelled with lower-case letters, based on a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test (p<0.0001). These assays were performed by JC and TD more than three times each in independent labs, with similar results. (G) Samples from the experiments shown in G were taken at 35 hours post infiltration (hpi) and subjected to western blot. Proteins are detected by anti-FLAG and anti-GFP antibodies; protein loading is indicated by Ponceau S staining. These assays were performed by JC three times with similar results.

Article Snippet: For auto-ubiquitination assays, 2 μg of recombinant E3 ligase and 0.075 μg of HA-tagged ubiquitin (R&D Systems; U-110) were incubated in 40 μL of reaction buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 25 mM KCl, 0.33 mM DTT, 1.5 mM ATP, 200 ng E1 (UBA1; AT2G30110), and 450 ng E2 (UBC8; AT5G41700) for the indicated times.

Techniques: Plasmid Preparation, Control, Transformation Assay, Construct, Binding Assay, Activation Assay, In Vitro, Ubiquitin Proteomics, Western Blot, Staining, Phospho-proteomics, Autoradiography, Expressing