ha ub k63 only 17606 plasmids  (Addgene inc)

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: IFI207 promotes antiviral responses by modulating STING ubiquitination and degradation

doi: 10.64898/2026.03.05.709838

Figure Lengend Snippet: Model of IFI207’s inhibition of STING degradation. STING is bound to IFI207, and after activation by DNA, dimerizes and moves from the ER to the Golgi. After the recruitment of TBK1 and STING phosphorylation, it normally becomes K63-ubiquitinated, binds HRS and is escorted to the endosome and then to the lysosome, where it is degraded. When bound to IFI207, K63 ubiquitination is inefficient, STING levels are higher, and STING downstream activation is increased, leading to a stronger type I IFN response. Created in BioRender. ENYA, T. ( 2025 ) https://BioRender.com/3yt291e .

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

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

doi: 10.1101/2025.04.08.647823

Figure Lengend Snippet: (A) Huh-7.5 cells were co-transfected with pCAG-Myc-LATS1, pRK5-HA-Ubiquitin, together with either pCAG-FLAG-Itch WT or pCAG-FLAG-Itch C868A. At 2 days post-transfection, the cells were harvested. The cell lysates were immunoprecipitated with anti-c-Myc antibody-conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed by immunoblotting. β-actin was used as a loading control. (B) Huh-7.5 cells were co-transfected with pCAG-Myc-LATS1, pRK5-HA−Ubiquitin, together with either pCAG-FLAG−WWP1 WT or pCAG-FLAG−WWP1 C890A. At 2 days post-transfection, cells were harvested. The cell lysates were immunoprecipitated with anti-c-Myc antibody−conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also immunoblotted, with β-actin serving as a loading control. (C) Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 1 and co-transfected with pCAG-Myc-LATS1 and pRK5-HA−Ubiquitin, together with either pCAG-FLAG-Itch or pCAG-FLAG−WWP1. Cell lysates were immunoprecipitated using anti-c-Myc antibody−conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed. β-actin was used as a loading control. (D) Huh-7.5 cells (3×10 5 cells per well in a 12-well plate) were transfected with 48 pmol of either control siRNA or Itch-specific siRNA. After 24 h, the cells were infected with HCV J6/JFH1 at an MOI of 2 and harvested at 2 days post-infection. The samples were analyzed by immunoblotting with the indicated antibodies. β-actin was used as a loading control. (E) Huh-7.5 cells (3×10 5 cells per well in a 12-well plate) were transfected with 48 pmol of either control siRNA or WWP1-specific siRNA. At 24 h after siRNA-transfection, the cells were infected with HCV J6/JFH1 at an MOI of 2. Cells were harvested at 2 days post-infection, and the samples were subjected to immunoblotting with the indicated antibodies. β-actin was used as a loading control.

Article Snippet: N-terminal HA-tagged ubiquitin (Ub) expression plasmids, including pRK5-HA−Ub-WT, pRK5-HA−Ub-K6, pRK5-HA−Ub-K11, pRK5-HA−Ub-K27, pRK5-HA−Ub-K29, pRK5-HA−Ub-K33, pRK5-HA−Ub-K48, and pRK5-HA−Ub-K63, were purchased from Addgene (Watertown, MA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Infection

Journal: bioRxiv

Article Title: HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

doi: 10.1101/2025.04.08.647823

Figure Lengend Snippet: Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 1, then co-transfected with pCAG-Myc-LATS1 and pRK5-HA-ubiquitin, together with pCAG-FLAG-Itch. After 2 days, the cells were harvested with or without pretreatment with JNK inhibitor SP6000125 (30 µM for 30 h), and the lysates were immunoprecipitated with anti-c-Myc antibody−conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed by immunoblotting. β-actin was used as a loading control.

Article Snippet: N-terminal HA-tagged ubiquitin (Ub) expression plasmids, including pRK5-HA−Ub-WT, pRK5-HA−Ub-K6, pRK5-HA−Ub-K11, pRK5-HA−Ub-K27, pRK5-HA−Ub-K29, pRK5-HA−Ub-K33, pRK5-HA−Ub-K48, and pRK5-HA−Ub-K63, were purchased from Addgene (Watertown, MA).

Techniques: Infection, Transfection, Immunoprecipitation, Western Blot, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: TRIM21 promotes type I interferon by inhibiting the autophagic degradation of STING via p62/SQSTM1 ubiquitination in systemic lupus erythematosus

doi: 10.3724/abbs.2025046

Figure Lengend Snippet: TRIM21 catalyzes K63-linked polyubiquitination of STING to inhibit STING autophagic degradation (A) HEK293T cells were transfected with either control vectors (Vec) or TRIM21 expression plasmids (TRIM21) and subsequently stimulated with 2′3′-cGAMP (2 μg/mL) for 0, 4, 8, or 24 h, and western blot analysis of STING and β-actin expressions were performed on cell lysates. HEK293T cells were transfected with Vec and subsequently stimulated with 2′3′-cGAMP (2 μg/mL) for 0, 4, 8, or 24 h, followed by treatment with MG132 (10 μM) or chloroquine (50 μM). Subsequently, western blot analysis was conducted on cell lysates to assess the expression levels of STING and β-actin. (C) HEK293T cells were transfected with TRIM21 expression plasmids and subsequently stimulated with 2′3′-cGAMP (2 μg/mL) for 0, 4, 8, or 24 h, followed by treatment with MG132 (10 μM) or chloroquine (50 μM). Cell lysates were then collected for western blot analysis of STING and β-actin. (D) Control, OE-TRIM21 and sh-TRIM21 HEK293T cells were stimulated with 2′3′-cGAMP (2 μg/mL) for 8 h and subsequently immunostained with anti-LC3 and DAPI. Scale bar: 50 μm.

Article Snippet: The HA-Ub (176462), Flag-p62 (204576), HA-Ub (K48) (17604), and HA-Ub (K63) (17606) plasmids were purchased from Addgene (Cambridge, USA).

Techniques: Transfection, Control, Expressing, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: TRIM21 promotes type I interferon by inhibiting the autophagic degradation of STING via p62/SQSTM1 ubiquitination in systemic lupus erythematosus

doi: 10.3724/abbs.2025046

Figure Lengend Snippet: TRIM21 catalyzes K63-linked polyubiquitination of p62/SQSTM1 and suppresses the interaction between p62/SQSTM1 and STING (A) sh-p62 and sh-TRIM21 or OE-TRIM21 were transfected into HEK293T cells, which were subsequently collected and lysed 24 h post-transfection for western blot analysis of p62/SQSTM1, TRIM21, STING, and β-actin. (B) HEK239T cells were transfected with either NC or overexpression TRIM21 (OE-TRIM21) plasmids. Following immunoprecipitation with anti-p62 or normal IgG, the lysates were subjected to immunoblotting using the specified antibodies. (C) HEK293T cells were transfected with NC or OE-TRIM21 plasmids, followed by stimulation with or without 2′3′-cGAMP (2 μg/mL) for 4 h. Subsequently, the cells were fixed, stained with anti-p62 antibody (green) and anti-STING antibody (red), and visualized by confocal microscopy. Scale bar: 50 μm. (D) HEK293T cells were transfected with plasmids encoding Flag-p62, HA-Ub, HA-Ub(K48), HA-Ub(K63), and Myc-TRIM21. Subsequently, after 24 h, the cells were subjected to ubiquitylation assay.

Article Snippet: The HA-Ub (176462), Flag-p62 (204576), HA-Ub (K48) (17604), and HA-Ub (K63) (17606) plasmids were purchased from Addgene (Cambridge, USA).

Techniques: Transfection, Western Blot, Over Expression, Immunoprecipitation, Staining, Confocal Microscopy, Ubiquitin Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: TRIM21 promotes type I interferon by inhibiting the autophagic degradation of STING via p62/SQSTM1 ubiquitination in systemic lupus erythematosus

doi: 10.3724/abbs.2025046

Figure Lengend Snippet: A working model for the positive regulation of the CGAS-STING signaling pathway by TRIM21 TRIM21 catalyzes K63-linked polyubiquitination of the selective autophagy receptor p62, which impairs its dimerization and cargo-sequestration functions, thereby preventing p62-STING interaction. This leads to defective autophagic degradation and cytosolic accumulation of STING, resulting in persistent activation of the cGAS-STING signaling pathway and subsequent aberrant production of type I interferons (IFN-I).

Article Snippet: The HA-Ub (176462), Flag-p62 (204576), HA-Ub (K48) (17604), and HA-Ub (K63) (17606) plasmids were purchased from Addgene (Cambridge, USA).

Techniques: Activation Assay

Journal: EMBO Reports

Article Title: OTUD6B regulates KIFC1-dependent centrosome clustering and breast cancer cell survival

doi: 10.1038/s44319-024-00361-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: HA-Ub K63 only , AddGene , #17606.

Techniques: Recombinant, Generated, Affinity Purification, Sequencing, Control, Negative Control, Cloning, Mutagenesis, CRISPR, Software, Microscopy, Real-time Polymerase Chain Reaction, Cell Analysis