prk5 ha ubiquitin k63 addgene  (Addgene inc)

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: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: (A) Immunoblot analysis using anti-ubiquitin to detect ubiquitination of endogenous ASC following its immunoprecipitation from WT or Peli1-KO BMDMs treated as indicated (upper). Cell lysates (input) were also subjected to immunoblot to detect expression of the indicated proteins (lower). (B) Ubiquitination analysis of endogenous ASC immunoprecipitated from Peli1 knockdown (using two different shRNA) or control iBMDMs, primed with LPS and further stimulated with nigericin for 45 min. (C) Ubiquitination analysis of exogenous ASC immunoprecipitated from Myc-ASC-transduced iBMDMs primed with LPS for 3 h and further stimulated for 45 min with nigericin and ATP or stimulated for 6 h with alum, poly dA:dT, flagellin, and MDP. (D–F) Ubiquitination analysis of exogenous ASC immunoprecipitated from 293T cells transfected with WT hemagglutinin (HA)-tagged ubiquitin (D) or WT and mutant forms of HA-ubiquitin (E and F) along with (+) the indicated expression vectors. (G) CoIP analysis of endogenous Peli1-ASC interaction in BMDMs primed with LPS (1 µg/mL) for 3.5 h with (+) or without (−) further stimulation for 30 min with nigericin. ASC complex was isolated by immunoprecipitation, and the precipitated ASC and Peli1 proteins were detected by immunoblot using anti-Peli1 and anti-ASC. (H) CoIP analysis of exogenous ASC and Peli1 or Peli1 mutants in transfected HEK293 cells transiently transfected with the indicated expression vectors. Data are representative of three independent experiments.

Article Snippet: pRK5-HA-Ubiquitin-K63 , Lim et al. J Neurosci. 2005 Feb 23. 25(8):2002-9. , Ted Dawson (Addgene plasmid # 17606).

Techniques: Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Expressing, Knockdown, shRNA, Control, Transfection, Mutagenesis, Isolation

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: (A) A diagram of human ASC with its lysine (K) residues and domains indicated. (B) A list of the six human ASC mutants harboring the indicated lysine-to-arginine substitutions. (C and D) 293T cells were transiently transfected with Myc-tagged human WT ASC or the six ASC mutants listed in (B), along with the indicated expression vectors. ASC and mutants were isolated by immunoprecipitation using anti-Myc and subjected to immunoblot with anti-HA to detect ASC ubiquitination. (E) Amino acid sequence alignment of ASC from different species, highlighting the conserved lysine residue K55. (F) Analysis of ubiquitin conjugation to mouse ASC WT and K55R mutant expressed in 293T cells along with (+) or without (−) HA-ubiquitin and E-tag-Peli1. Data are representative of three independent experiments.

Article Snippet: pRK5-HA-Ubiquitin-K63 , Lim et al. J Neurosci. 2005 Feb 23. 25(8):2002-9. , Ted Dawson (Addgene plasmid # 17606).

Techniques: Transfection, Expressing, Isolation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Sequencing, Residue, Conjugation Assay, Mutagenesis

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pRK5-HA-Ubiquitin-K63 , Lim et al. J Neurosci. 2005 Feb 23. 25(8):2002-9. , Ted Dawson (Addgene plasmid # 17606).

Techniques: Transfection, Staining, Activity Assay, Plasmid Preparation, Ubiquitin Proteomics, Software

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