479 ha ub k48  (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: 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